Stabilized anthocyanin compositions

ABSTRACT

The invention describes stabile anthocyanin compositions, methods to prepare such compositions and also methods of use of such compositions to treat various afflictions. The present invention describes unique compositions of an anthocyanin and a stabilizing compound such that the combination of the two components provides that the anthocyanin does not readily undergo degradation, such as oxidation, pH instability, etc.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims benefit of U.S. Ser. No. 60/895,034 filed onMar. 15, 2007, U.S. Ser. No. 60/952,113 filed Jul. 26, 2007, and U.S.Ser. No. 60/985,603 filed Nov. 5, 2007, the contents of each of whichare incorporated in their entirety herein by reference.

FIELD OF THE INVENTION

The invention relates generally to methods and compositions useful tostabilize anthocyanins and anthocyanidins.

BACKGROUND OF THE INVENTION

Anthocyanins are water soluble pigments which are responsible for theattractive colors of many flowers, fruit and leaves. Generally, they canbe extracted from plants by acidified alcoholic solvents and many areavailable commercially as food colorants. They are often supplied withmalto dextrin as a diluent in a concentration suitable for inclusion inbeverages or other foods such as cereals.

Anthocyanidines, the aglyconic component of anthocyanins, have a basicstructure as shown in Formula I.

Typical examples are: cyanidin (hydroxylated at positions 3, 5, 7, 3′,4′), delphinidin (hydroxylated at positions 3, 5, 7, 4′, 5′) andpelargonidin (hydroxylated at positions 3, 5, 7, 3′). The hydroxylgroups are usually glycosylated (e.g., an anthocyanin) and/ormethoxylated (e.g. malvidin is substituted at the 3′ and 5′ hydroxylgroups and paeonidin and petunidin are substituted at the 3′ hydroxylgroup).

Anthocyanins are water-soluble glycosides of polyhydroxyl andpolymethoxyl derivatives of 2-phenylbenzopyrylium or flavylium salts.Individual anthocyanins differ in the number of hydroxyl groups presentin the molecule, the degree of methylation of these hydroxyl groups, thenature, number and location of sugars attached to the molecule and thenumber and the nature of aliphatic or aromatic acids attached to thesugars in the molecule. Hundreds of anthocyanins have been isolated andchemically characterized by spectrometric tools. Cyanidins and theirderivatives are the most common anthocyanins present in vegetables,fruits and flowers.

Anthocyanins share a basic carbon skeleton in which hydrogen, hydroxylor methoxyl groups can be found in six different positions as notedabove. In fruits and vegetables, six basic anthocyanin compoundspredominate, differing both in the number of hydroxyl groups present onthe carbon ring and in the degree of methylation of these hydroxylgroups. The identity, number and position of the sugars attached to thecarbon skeleton are also variable; the most common sugars that can belinked to carbon-3, carbon-S and, sometimes, carbon-7, are glucose,arabinose, rhamnose or galactose. On this basis, it is possible todistinguish monosides, biosides and triosides.

Another important variable that contributes to the chemical structure ofanthocyanins is the acylating acid that can be present on thecarbohydrate moiety. The most frequent acylating agents are caffeic,ferulic, sinapic and p-coumaric acids, although aliphatic acids such asacetic, malic, malonic, oxalic and succinic acids can also occur. Up tothree acylating acids can be present simultaneously.

Due to their particular chemical structure, anthocyanins andanthocyanidins are characterized by an electron deficiency, which makesthem very reactive toward reactive oxygen species (ROS), also known asfree radicals; they are consequently considered to be powerful naturalantioxidants.

Anthocyanins, due in part to the nature of their chemical structure,tend to be unstable and susceptible to degradation. Additionally, thestability of anthocyanins is effected by pH, storage over a period ofmonths, storage temperature, presence of enzymes, light, oxygen, and thepresence of proteins, flavonoids and minerals

More particularly, the bioavailability of anthocyanins is low due totheir sensitivity to changes in pH. Anthocyanins are generally stable atpH values of 3.5 and below, and are therefore stable under stomachconditions. However, they degrade at higher pH values, such as thosemore typical for the intestinal tract (pH of 7) and thus beneficialabsorption and nutritional value is greatly reduced.

Therefore, a need exists for a composition and/or method that providesstabilized anthocyanins.

BRIEF SUMMARY OF THE INVENTION

The present invention surprisingly provides stabile anthocyanincompositions, methods to prepare such compositions and also methods ofuse of such compositions to treat various afflictions. The presentinvention provides unique compositions of an anthocyanin and astabilizing compound such that the combination of the two componentsprovides that the anthocyanin does not readily undergo degradation. Upuntil the time of the invention, it was known that anthocyanins woulddegrade upon exposure to environmental stresses, such as air, light,proteins, or enzymes. More troublesome was the instability ofanthocyanins in solutions having a neutral or basic pH.

Surprisingly, the present invention provides that use of cysteine incombination with an anthocyanin composition (whether it be ananthocyanidine or an anthocyanoside) helps to increase the delivery ofthe anthocyanin to a subject in need thereof by at least twice theamount relative to a subject that ingests an anthocyanin compositionwithout the presence of cysteine. It has been surprisingly found thatplasma concentration levels of the anthocyanin where the anthocyanin isdelivered in the presence of cysteine after 4 hours is at least twicethe plasma concentration of an anthocyanin delivered without thecysteine. Therefore, the present invention provides a method to increasethe amount of bioavailable anthocyanin in a subject by administering tothe subject an effective amount of an anthocyanin and cysteine. Theadministration can be by any means, but oral delivery is generallypreferred. In one embodiment, the ratio of the anthocyanin to thecysteine is about 10 to about 1, on a weight basis.

In one aspect, the present invention provides a stabilized anthocyaninextract composition that includes an anthocyanin extract and astabilizing compound having at least one —SH group. Suitable examples ofstabilizing compounds include (reduced) glutathione, dihydrolipoic acid,cysteine, yeast extract and mixtures thereof.

Although there are literally thousands of anthocyanin extracts, all ofwhich should be considered included within the realm of thisspecification, suitable examples of anthocyanin extracts of particularinterest include bilberry extract, blackcurrant extract, cranberryextract, black soybean extract, cowberry extract, blueberry extract andmixtures of two or more thereof.

In one aspect, the ratio of stabilizing compound to anthocyanin extractis about 0.1 to about 10, more particularly about 0.5 to about 5, andmore particularly about 1 to about 1.

In another aspect, the stabilized anthocyanin extract composition isstabile toward degradation when exposed to an aqueous environment with apH of about 2 or greater, such as a pH of about 3, of about 4, of about5, pH of about 6, pH of about 7 pH of about 8, of about 9, of about 10,or about 11, of about 12 or even higher, e.g. 14.

In still another aspect, the stabilized anthocyanin extract is ananthocyanoside.

In still yet another aspect, the stabilized anthocyanin extract is ananthocyanidin.

In still other aspects of the invention, the stabilized anthocyaninextract includes one or more anthocyanosides that are glycosides ofperlargonidin, peonidin, cyanidin, malvidin, petunidin, delphinidin.

The present invention also pertains to methods of preparing thestabilized anthocyanin compositions described herein.

The present invention further pertains to methods of treatment ofvarious ailments by administration of a therapeutically effective amountof the stabilized anthocyanin compositions described herein.

Therefore, the present invention further provides bioavailablestabilized anthocyanin compositions.

While multiple embodiments are disclosed, still other embodiments of thepresent invention will become apparent to those skilled in the art fromthe following detailed description. As will be apparent, the inventionis capable of modifications in various obvious aspects, all withoutdeparting from the spirit and scope of the present invention.Accordingly, the detailed descriptions are to be regarded asillustrative in nature and not restrictive.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides evidence of a lack of bathochromic shift forcy-3-O-glucoside in a 1 mMolar solution of glutathione or DHLA.

FIG. 2 provides evidence of a lack of hyperchromic shift forcy-3-O-glucoside in a 1 mMolar solution of glutathione or DHLA.

FIG. 3 provides percent residual anthocyanosides of bilberry extract(unprotected).

FIG. 4 provides percent residual anthocyanosides of bilberry extractthat is DHLA protected in contrast to unprotected seen in FIG. 3.

FIG. 5 provides percent residual anthocyanosides of bilberry extractthat is GSH protected in contrast to unprotected seen in FIG. 3.

FIGS. 6A through 6D provide comparative degradation kinetics of bilberryextract that is DHLA-protected.

FIG. 7 provides comparative degradation kinetics ofdelphinidin-3-O-galactoside that is unprotected or protected withglutathione.

FIG. 8 provides comparative degradation kinetics ofpetunidin-3-O-galactoside that is unprotected or protected withglutathione.

FIG. 9 provides comparative degradation kinetics ofdelphinidin-3-O-galactoside that is unprotected or protected withglutathione.

FIG. 10 provides comparative degradation kinetics ofcyanidin-3-O-galactoside at two pH values with and without protectionwith glutathione.

FIG. 11 provides comparative degradation of 15 bilberry anthocyanosidesthat are DHLA protected.

FIG. 12 provides comparative degradation of 15 bilberry anthocyanosidesthat are glutathione (GSH) protected.

FIG. 13 demonstrates stability in buffered solution of leadanthocyanoside (Black current) in the presence of glutathione at a pH of7 at 37° C. over time.

FIG. 14 demonstrates the stability in incubation medium of leadanthocyanoside (Black current) in the presence of glutathione withCaCo-2 cells at a pH of 7 at 37° C. over time.

FIG. 15 demonstrates cellular uptake of lead-anthocyanosides into CaCo-2cells.

FIG. 16 provides degradation of bilberry anthocyanosides with/withoutglutathione (1 hour at 37° C., pH=7.0).

FIG. 17 demonstrates cellular uptake of bilberry anthocyanosideswith/without glutathione into CaCo-2 cells.

FIG. 18 provides the gradient profile for HPLC analysis of the CaCo-2experiments.

FIG. 19 is a graphical representation of the residual ratio and pHvalues over a pH range of 3 to 11 for the stability of bilberry extracttreated with GSH and instability of bilberry extract not treated withGSH (at the same pH values).

FIG. 20 is a graphical representation of the protective effect of GSHover a pH range.

FIG. 21 is an HPLC chromatogram of fresh solution of 20070601, timezero.

FIG. 22 is an HPLC chromatogram of 20070601 after 4 hours at 37° C.

FIG. 23 is an HPLC chromatogram of fresh solution of 20070602, timezero.

FIG. 24 is an HPLC chromatogram of 20070602 after 4 hours at 37° C.

FIG. 25 provides HPLC peak comparisons of 20070601 at time zero andafter 4 hours at 37° C.

FIG. 26 provides HPLC peak comparisons of 20070602 at time zero andafter 4 hours at 37° C.

FIG. 27 provides a comparison of anthocyanidins observed in human plasmaafter treatment with bilberry extract or a bilberry/cysteine combinationof the invention.

FIGS. 28 a and 28 b provides a comparison of total cyandin anddelphinidin, respectively, observed in human plasma after treatment withbilberry extract or a bilberry/cysteine combination of the invention.Upper dotted line is bilberry/cysteine combination; lower hashed line isbilberry extract only.

FIG. 29 provides a comparison of total petunidin observed in humanplasma after treatment with bilberry extract or a bilberry/cysteinecombination of the invention. Upper dotted line is bilberry/cysteinecombination; lower hashed line is bilberry extract only.

FIG. 30 provides a comparison of total peonidin observed in human plasmaafter treatment with bilberry extract or a bilberry/cysteine combinationof the invention. Upper dotted line is bilberry/cysteine combination;lower hashed line is bilberry extract only.

FIG. 31 provides a comparison of total malvidin observed in human plasmaafter treatment with bilberry extract or a bilberry/cysteine combinationof the invention. Upper dotted line is bilberry/cysteine combination;lower hashed line is bilberry extract only.

FIG. 32 provides comparative C_(max) observed on day 1 for cyandin,delphinidin, petunidin, peonidin and malvidin for bilberry extractversus a bilberry extract/cysteine combination.

FIG. 33 provides comparative C_(max) observed on day 7 for cyandin,delphinidin, petunidin, peonidin and malvidin for bilberry extractversus a bilberry extract/cysteine combination.

FIG. 34 provides comparative AUC_(0-inf) observed on day 1 for cyandin,delphinidin, petunidin, peonidin and malvidin for bilberry extractversus a bilberry extract/cysteine combination.

FIG. 35 provides comparative AUC_(0-inf) inf observed on day 7 forcyandin, delphinidin, petunidin, peonidin and malvidin for bilberryextract versus a bilberry extract/cysteine combination.

FIG. 36A provides total cyanidin plasma levels in plasma at 4 hours, 1day, 7 days and on day 4, the two points represent 0 and 0.5 hoursrespectively for bilberry extract without cysteine.

FIG. 36B provides total cyanidin plasma levels in plasma at 4 hours, 1day, 7 days and on day 4, the two points represent 0 and 0.5 hoursrespectively for bilberry extract with cysteine.

FIG. 37A provides total delphinidin plasma levels in plasma at 4 hours,1 day, 7 days and on day 4, the two points represent 0 and 0.5 hoursrespectively for bilberry extract without cysteine.

FIG. 37B provides total delphinidin plasma levels in plasma at 4 hours,1 day, 7 days and on day 4, the two points represent 0 and 0.5 hoursrespectively for bilberry extract with cysteine.

FIG. 38A provides total petunidin plasma levels in plasma at 4 hours, 1day, 7 days and on day 4, the two points represent 0 and 0.5 hoursrespectively for bilberry extract without cysteine.

FIG. 38B provides total petunidin plasma levels in plasma at 4 hours, 1day, 7 days and on day 4, the two points represent 0 and 0.5 hoursrespectively for bilberry extract with cysteine.

FIG. 39A provides total peonidin plasma levels in plasma at 4 hours, 1day, 7 days and on day 4, the two points represent 0 and 0.5 hoursrespectively for bilberry extract without cysteine.

FIG. 39B provides total peonidin plasma levels in plasma at 4 hours, 1day, 7 days and on day 4, the two points represent 0 and 0.5 hoursrespectively for bilberry extract with cysteine.

FIG. 40A provides total malvidin plasma levels in plasma at 4 hours, 1day, 7 days and on day 4, the two points represent 0 and 0.5 hoursrespectively for bilberry extract without cysteine.

FIG. 40B provides total malvidin plasma levels in plasma at 4 hours, 1day, 7 days and on day 4, the two points represent 0 and 0.5 hoursrespectively for bilberry extract with cysteine.

DETAILED DESCRIPTION

The present invention relates to compositions containing one or moreanthocyanins and one or more stabilizing compounds. The compositions arethus “stabile” in that the anthocyanin does not readily degrade over agiven period of time.

In the specification and in the claims, the terms “including” and“comprising” are open-ended terms and should be interpreted to mean“including, but not limited to . . . . ” These terms encompass the morerestrictive terms “consisting essentially of” and “consisting of.”

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural reference unless thecontext clearly dictates otherwise. As well, the terms “a” (or “an”),“one or more” and “at least one” can be used interchangeably herein. Itis also to be noted that the terms “comprising”, “including”,“characterized by” and “having” can be used interchangeably.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs. All publications and patentsspecifically mentioned herein are incorporated by reference in theirentirety for all purposes including describing and disclosing thechemicals, instruments, statistical analyses and methodologies which arereported in the publications which might be used in connection with theinvention. All references cited in this specification are to be taken asindicative of the level of skill in the art. Nothing herein is to beconstrued as an admission that the invention is not entitled to antedatesuch disclosure by virtue of prior invention.

In one aspect, the present invention provides a stabilized anthocyanincomposition that includes an anthocyanin extract and a stabilizingcompound having at least one —SH group.

The term “anthocyanin” as used herein is intended to include bothglycosylated anthocyanins (anthocyanosides) as well as the aglycon ofthe anthocyanoside (anthocyanidin). Throughout the specification,reference to the aglyconic anthocyanidin will often be made but shouldin no way be construed as limiting unless otherwise noted. Whereineither term is used, unless otherwise noted, the terms are usedinterchangeably and are intended to include the glycosylated as well asaglyconic materials.

Anthocyanidines, the aglyconic component of anthocyanins, have a basicstructure as shown in Formula II

Formula II

Antho- cyanidin R₁ R₂ R₃ R₄ R₅ R₆ R₇ Auantinidin H OH H OH OH OH OHCyanidin OH OH H OH OH H OH Delphinidin OH OH OH OH OH H OH EuropinidinOCH₃ OH OH OH OCH₃ H OH Luteolinidin OH OH H H OH H OH Pelargonidin H OHH OH OH H OH Malvidin OCH₃ OH OCH₃ OH OH H OH Peonidin OCH₃ OH H OH OH HOH Petunidin OH OH OCH₃ OH OH H OH Rosinidin OCH₃ OH H OH OH H OCH₃

where R₁ through R₇ provide representative examples of anthocyanidins.

The glycosylated forms of anthocyanins are more water soluble and stablethan anthocyanidins. Anthocyanosides are classified by the number ofglycosyl units they contain. Monoglycosides include one saccharidicmoiety, which is primarily attached to the 3-hydroxyl group of theaglycon. Diglycosides generally contain two monosaccharides at the 3 and5 hydroxy positions and occasionally at the 3 and 7 hydroxyl positions.Triglycosides have attachment generally where there are two units at the3 position and one at the C-5 or C-7 position. Glycosylations at the 3′,4′ and/or 5′ positions are also possible.

The most common sugars of anthocyanins include the monosaccharidesglucose, rhamnose, galactose, arabinose and xylose. The di- andtrisaccharides found most often in anthocyanins are rutinose, sophorose,sambubiose and glucorutinose.

Anthocyanins can be acylated through one or more hydroxyls with acarboxylic acid. The acids are most commonly linked to the 6 position ofthe monosaccharide but the 2, 3 and 4 positions of the monosaccharidesare also possible. Common aliphatic acids include malonic, acetic,malic, succinic, and oxalic acids. Common aromatic phenolic acids andaliphatic dicarboxylic acids include coumaric, acffeic, sinapic,ferulic, oxalic, malonic, malic, succinic and gallic acid.

The term “extract” is intended to mean anthocyanin materials obtainedfrom plant sources, such as leaves, twigs, bark, roots, stem, seeds,flowers, berries, fruit, for example, by routine isolation methods fromsuitable plants sources noted, but not limited to, those describedherein. There are various methods for the extraction of anthocyaninsknown to those of skill in the art. Some of these methods are describedin, for example, U.S. Pat. No. 5,817,354; U.S. Pat. No. 5,200,186; U.S.Pat. No. 5,912,363; U.S. Pat. No. 4,211,577; U.S. Pat. No. 4,302,200(each incorporated herein by reference).

Examples of suitable anthocyanin-containing plants include, but are notlimited to, fruits, vegetables, flowers and other plants selected fromthe group consisting of Acer macrophyllum, Acer platanoides, acerola,Ajuga reptans, apple, apricot, Artict bramble, avocado, banana,barberry, barley, Begonia semperfiorens, Bellis perennis, Bletillastriata, bilberry, black beans, black soybeans, black, blue and purplepotatoes, blackberry, blueberry, bog whortleberry, boysenberry,buckwheat, cacao, Camellia sinensis, canarygrass, Caucasian blueberry,Chimonanthus praecox, celery, Cerasus avium, cherry, cherry laurel,chicory, chive, chokeberry, Cornelian cherry, cornflower, cotoneaster,cowberry, cranberry, crowberry, chrysanthemum, Cynomorium coccineum,Dahlia variabilis, danewort, deerberry, Dendrobium, dwarf dogwood,Echinacea purpea, eggplant, elderberry, fababean, Fatsia japonica,feijoa, fig, garlic, gerbera, ginseng, Globe artichoke, gooseberry,grapes, guava, hawthorn, hibiscus or roselle, Hibiscus Sabdaiffa,highbush blueberry, hollyhock, honeysuckle, Ipomoea purpurea, Irisensata, Java plum, Jerusalem artichoke, kokum, Laeliocattleya, lentil,loganberry, lupine, lychee, maize, mango, mangosteen, maqui, Matthiolaincana, meconopsis, Metrosideros excelsa, millet, mountain ash berry,mulberry, myrtle berry, olive, onion, orange, ornamental cherry, passionfruit, pea, peach, peanut, pear, perilla, petunia, Phalaenopsis, Phalsa,Pharbitis, Pineapple, pistachio, plum, pomegranate, Phragmitesaustralis, purple carrot, quince, rabbiteye blueberry, radish, red andblack currant, red and black raspberry, red cabbage, rice, rhubarb,rosehip, rye, saffron, sarracenia, sheepberry, Sophronitis coccinea,sorghum, sparkleberry, strawberry, Fragada Vesca, sugarcane, sunflower,sweet cherry, sweet potato, tamarillo, tamarind, taro, tart cherry,Tulip greigii, turip, water lily, Weigela, wheat, wild rice, Verbenahybrida, yam and mixtures thereof.

Although there are literally thousands of anthocyanin extracts, all ofwhich should be considered included within the realm of thisspecification, suitable examples of anthocyanin extracts of particularinterest include bilberry extract, blackcurrant extract, cranberryextract, black soybean extract, cowberry extract, blueberry extract andmixtures of two or more thereof.

Typically the extract is concentrated by various methods to provide asolution enriched in anthocyanins. For example, ultrafiltration can beused to remove unwanted components by molecular weight cut offs. Theretentate from the filtration can be stored as a liquid or, for example,can then be further concentrated into a powder by spray drying, freezedrying, flash drying, fluidized bed drying, ring drying, tray drying,vacuum drying, radio frequency drying or microwave drying. Ultimately,the extract should contain at least 10% by weight anthocyanin content.Commercially available anthocyanins can be obtained from sources such asArtemis International, Fort Wayne, Ind. Commercially obtainedanthocyanin extracts should contain at least 10% by weight anthocyanincontent. The extracts, therefore, contain anthocyanin(s) and other plantmaterials such as other flavinoids, sugars, etc.

Anthocyanin extracts can be further purified by one or more methodsknown in the art, such as chromatography, gel chromatography, highperformance liquid chromatography, crystallization, affinitychromatography, partition chromatography and the like. Identification ofthe particular anthocyanin(s) can be accomplished by methods know tothose skilled in the art and include ¹H NMR, chemical degradation,chromatography and spectroscopy, especially homo- and heteronucleartwo-dimensional NMR techniques for the characterization of the isolatedanthocyanin compounds.

The term “purified” or “isolated” is used in reference to thepurification and/or isolation of one or more anthocyanins from ananthocyanin extract as described above. Again using conventional methodsknown in the art, various components of the anthocyanin extract can beseparated into purified materials. In one aspect of the invention, theanthocyanin(s) of the extract are substantially purified and isolated bytechniques known in the art. The purity of the purified compounds isgenerally at least about 90%, preferably at least about 95%, and mostpreferably at least about 99% and even more preferably at least about99.9% (e.g. about 100%) by weight.

In accordance with the present invention, the anthocyanin extractcontains one or more anthocyanins and/or anthocyanidins selected fromthe group consisting of peonidin, cyanidin, pelargonidin, delphinldin,petunidin, malvidin, apigenindin, auratinidin, capensinidin,europinidin, hirsutidin, 6-hydroxycyanidin, luteolinidin,5-methylcyanidin, pulchellidin, rosinidin, tricetnidin, derivatives andmixtures thereof. In one embodiment, the anthocyanins and anthocyanidinsare selected from the group consisting of cyanidin, peonidin, malvidin,petunidin, delphinidin, their glycoside derivatives, and mixturesthereof. In yet another embodiment, the extract contains at least onecyanidin-based anthocyanin.

Anthocyanins that can be useful in the inventions described hereininclude, but are not limited to, cyanidin-3-glucoside; cyanidin3-glucosylrutinoside; cyanidin-3-gentibioside; cyanidin-3-rutinoside,cyanidin-3-sambunigrin, cyanidin-3-samb-5-glucoside,cyanidin-3-galactoside, peonidin-3-rutinoside, peonidin-3-glucoside,peonidin-3-galactoside, peonidin, cyanidin, cyanidin-3 sophoroside,pelargonidin, delphinidin, delphinidin-3-glucoside,delphinidin-3-galactoside, petunidin, petunidin-3-glucoside,petunidin-3-galactoside, malvidin, malvidin-3-arabinoside,malvidin-3-glucoside, malvidin-3-galactoside, kaempferol, hesperidin,gentiodelphin, platyconin, cinerarin and the like.

Suitable examples of anthocyanins from various plants, include, but arenot limited to Acer macrophyllum, Cyanidin derivative, Acer platanoides,Cyanidin 3-(2″,3″-digalloyl-beta-glucopyranose (3%), Cyanidin3-(2″-galloyl-beta-glucopyranose (37%), Cyanidin 3-beta-glucopyranoside(60%), Acerola, Malpighia marginata, Cyanidin-3-glucoside,Cyanidin-3-glucoside, Ajuga reptans, Cyanidin 3-(di-p-coumaroyl)sophoroside-5-glucoside, Apple, Malus spp, Cyanidin 3-galactoside,Cyanidin 3-galactoside, Cyanidin 3-arabinoside, Cyanidin 3-glucoside,Cyanidin 3arabinoside, Cyanidin 3-xyloside, Cyanidin 3glucoside,Cyanidin 3-xyloside, Apricot, Prunus armeniaca, Cyanidin-3-glucoside,Cyanidin-3glucoside, Artic bramble, Rebus spp, Avocado, Persea spp,Acylated cyanidin 3,5-diglucoside, Cyanidin 3-galactoside, Cyanidin3-galactoside, Banana, Musa acuminata, M. balbisiana, Barberry, Berberisspp., Cyanidin-glucoside, Cyanidin-glucoside, Barley, Hordeum vulgare,Cyanidin and cyaniding glycosides, Bean, Pheseolus vulgaris (severalcultivars), Cyanidin 3-glucoside, Cyanidin 3-glucoside, Cyanidin3,5-diglucoside, Begonia semperflorens cvs, Cyanidin derivative,Benibana-cha, Camellia sinensis, Cyanidin 3-O-beta-D galactoside,Cyanidin 3-O-beta-D-galactoside, Bellis perennis, 3 Cyanidin3-derivatives, Bletilla striata, Acylated cyanidin 3,7,3′-triglucosidederivatives, Bilberry, Vaccinium myrtillus, Artemis/Iprona; Indena,Cyanidin-3-gal actoside (22%); Cyanidin-3-galactoside,Cyanidin-3-glucoside (9%), Cyanidin-3glucoside, Black beans, Phaseolus,Cyanidin-3-glucoside (96%), Cyanidin-3glucoside, Blackberry (Europeanand American), Moriferi veri, Rubus caesius, R. Alleghniensis, R.argufus, R. cuneifolius, R. setosus, R. trivials, Cyanidin-glucoside(70-100%), Cyanidin-glucoside, Cyanidin-rutinoside, Black grapes, Manyvarieties, Black potatoes, Solanumtuberosum tuberosum,Cyanidin-glycosides, Black raspberry, Rubus occidentalis,Cyanidin-sambubloside (20%); Cyanidin-sambubloside,Cyanidin-xylosylrutinoside (40%); Cyanidin-glucoside,Cyanidin-glucoside, (17%), Cyanidin-rutinoside (23%), Black soybeans,Glycine max, Cyanidin-3-glucoside (96%), Cyanidin-3-glucoside,Blueberries, Five common Vaccinium spp, Cyanidin-glucoside (3%);Cyanidin-glucoside, Cyanidin-galactoside (3%), Cyanidin galactoside,Cyanidin-arabinoside (3%), Cyanidin-3-arabinoside, Bog whortleberry,Vaccinium uliginosum, Cyanidin-3-glucoside (14%), Cyanidin 3 glucoside(14%), Cyanidin #arabinoside (10%), Cyanidin-3-arabinoside (10%),Cyanidin 3-galactoside (6.5%), Cyanidin-3-galactoside (6.5%),Boysenberry, new Zealand, Cyanidin-3-sophoroside (44.5%),Cyanidin-3-glucoside, Cyanidin-3-glucoside (26.4%), Cyanidin-3glycosylrutinoside (25.8%), Cyanidin-rutinoside (3.3%), Buckwheat,Fagopyrum species, Cyanidin-3-glucoside, Cyanidin-3-glucoside, Cyanidin3-galactoside, Cyanidin-3-galactoside, Cacao, Theobroma cacao, Cyanidin3-glucoside (suspected), Cyanidin-3-glucoside (suspected), Celery, Apiumspp, Cherry laurel, Prunus laurocerasus, Cyanidin-3-arabinoside,Cyanidin-3-arabinoside, Chicory, Cichorium intybus, Cyanidin3-glucoside, Cyanidin 3-glucoside, Chive, Allium schoenoprasum,Cyanidin-3-glucoside, Cyanidin-3-glucoside, Cyanidin-3-acetylglucoside,Cyanidin 3-(6 malonylglucoside), Cyanidin 3-(3,6 dimalonylglucoside),Chokeberry, Aronia melanocarpa, Artemis/Iprona, Cyanidin-3-galactoside(64.5%), Cyanidin-3-galactoside, Cyanidin-3-arabinoside (28.9%),Cyanidin-3 arabinoside, Cyanidin-3-glucoside (2.4%), Cyanidin-3glucoside, Cyanidin-3-xyloside (4.2%), Cyanidin-3-xyloside, Coffee,Coffea arabica cv. Bourbon Vermelho, Cyanadin-3-glycoside, Cyanadin3,5-diglyeoside, Cyanadin 3-glycoside, Cotoneaster, Cotoneaster Medic.Spp, Cyanidin 3-glucoside, Cyanidin 3-glucoside, Cyanidin 3-galactoside,Cyanidin 3-rutinoside, Cyanidin 3 galactoside, Cowberry or Lingonberry,V. vitisidaea, Cyanidin 3-galactoside Cyanidin 3-arabinoside, Cyanidin3-galactoside, Cyanidin 3-glucoside, Cyanidin 3 arabinoside, Cyanidin 3glucoside, Chimonanthus praecox, Cyanidin 3-O-glucoside,Cyanidin-3-O-glucoside, Acylated cyanidin 3-O-glucoside, Cyanidinglycoside, Cranberry (American and European), Vaccinium macrocorpon,Ocean Spray, Cyanidin-galactoside (16-24%), Cyanidin-galactoside, V.oxycoccus, Cyanidin-arabinoside (13-25%), Cyanidin arabinoside,CrOwberry, Empetrum nigrum, Cyanidin 3-glucoside Cyanidin3,5-diglucoside, Cyanidin 3-glucoside, Cyanidin 3-rutinoside, Cyanidin3-sophoroside, Chrysanthemum, Dendranthema Grandiflorum, Cyanidin3-O-(6′-O-malonyl-beta-glucopyranoside, Currant (red and black), Ribesrubrum, R. nigrum, Cyanidin-glucoside (2-10%), Cyanidin-glucoside,Cyanidin sambubioside, Cyanidin-rutinoside (8-17%),Cyanidin-sambubioside (9-31%), Cyanidin-sophoroside (4-9%), Cyanidinxylosylrutinoside (28-73%), Cyanidin glucosylrutinoside (14-28%),Cyneinonurn coccineum, Cyanidin 3-O-glucoside (92%), Cyanidin3-O-glucoside (92%), Cyanadin 3-O-(6-O-rhamnosylglucoside (8%),Danewort, Sambucus ebulus, Cyanidin 3-xylosylglucoside, Cyanidin3-sambubioside, Cyanidin 3 sambubloside, Cyanidin 3-glucoside, Cyanidin3-sambubioside-5-glucoside, Cyanidin 3,5 diglucoside, Cyanidin3-glucoside, Cyanidin 3-arabinoglucoside, Dendrobium, Phalaenapsis spp,Cyanidin derivatives, Dwarf dogwood, Comus suecica, Cyanidin 3-glucoside(4%), Cyannidin 3-glucoside (4%), Cyanidin 3-galactoside (16%), 2Cyanidin derivatives (80%), Echinacea, Echinacea spp., Eldenberry,Sambucus nigra, Artemis/Iprona, Cyanidin-3-glucoside (42%),Cyanidin-3-glucoside, Cyanidin-3-sambubioside (43%)Cyanidin-3,5-diglucoside (2%), Cyanidin-3 sambubloside-5 glucoside (9%),Gentians spp, Cyanidin 3-O-beta-D-glucoside and 3 other derivatives,Cyanidin 3-O-beta-D-glucoside, Fatsia japonica, Cyanidin 3-lathyroside,Feijoa, Feijoa sellowiana, Cyanidin 3-glucoside, Cyanidin 3-glucoside,Fig, Ficus carica spp, Cyanidin 3-rhamnoglucoside, Cyanidin3,5-diglucoside, Cyanidin 3-glucoside, Forsythia X, intermedia cv,Spring Glory, Cyanidin derivatives, Garlic, Allium sativum, Cyanidin3-glucoside, Cyanidin 3-glucoside, Cyanidin 3-glucoside, Cyanidin3-glucoside monoacylated, Cyanidin 3-glucoside triacylated, Ginseng,Panax ginseng, Panax quinquefolius, Cyanidin3-O-β-D-xylopyranyl-(12)-β-D-glucopyranoside, Globe artichoke, Cynarascolymus, Cyanidin 3-caffeylglucoside, Cyanidin 3-caffeylsophoroside,Cyanidin 3-dicaffeylsophoroside, Gooseberry, Ribes spp, Cyanidin3-glucoside, Cyanidin 3-glucoside, Cyanidin 3-rutinoside, Grape, Vinisvinifera, Cyanidin 3-monoglucoside, Cyanidin 3-monoglucoside, Cyanidin3-monoglucoside-acetate, Cyanidin 3-monoglucoside-p-coumarate, Guava,Psidium guajavica, Cyanidin 3-glucoside, Cyanidin 3-glucoside, Hawthorn,Crataegus spp, Cyanidin 3-galactoside, Cyanidin 3-galactoside, Cyanidin3-arabinoside, Cyanidin 3-glucoside, Cyanidin 3 glucoside, Hibiscus orRoselle, Hibiscus sabdariffa, Cyanidin-sambubioside (30%), Hollyhock,Althaea rosea, Cyanidin 3-glucoside, Cyanidin 3-rutinoside, Cyanidin3-glucoside, Other cyaniding glucosides, Honeysuckle, Lonicera nitida,Cyanidin 3-rutinoside, Cyanidin 3-glucoside, Cyanidin 3-glucoside,Japanese garden iris, Iris ensata, Cyanidin 3RG, Cyanidin 3RG5G,Cyanidin 3Rgac5G, Ipornoea purpurea, Six acylated cyanidin3-sophoroside-5 glucosides, Java plum, Mytciana jaboticaba, Cyanidin3-glucoside, Cyanidin 3-glucoside, Jerusalem artichoke, Helianthustuberosus, Kokum, Garcinia indica, Cyanidin 3-glucoside, Cyanidin3-glucoside, Cyanidin 3-sambubioside, Cyanidin 3-sambubioside,Laelioeattleya cv Mini purple, Acylated cyaniding derivatives, Lactucasaliva, Cyanidin 3-O-(6″-malonylglucoside), Loganberry, Rubusloganbaccus, Cyanidin-sophoroside (48.1%), Cyanidin-glucoside,Cyanidin-glucoside (21.6%), Cyanidin-rutinoside (6.2%), Lupine, Lupinusspp, Cyanidin glycosides, presence confirmed, Lychee, Litchi chinensis,Cyanidin 3-glucoside, Cyanidin 3-glucoside, Cyanidin 3-galactoside,Cyanidin 3-rutinoside, Cyanidin 3 galactoside, Maize, Zea mays, Cyanidin3-glucoside, Cyanidin 3-glucoside, Cyanidin 3-(6″-malonylglucoside)Cyanidin 3(3″,6″dimalonyl-glucoside) Mango, Mangifera indica, (Cyanidinglycosides, Mangosteen, Garcina mangostana, Cyanidin 3-sophoroside,Cyanidin 3-glucoside, Cyanidin 3-glucoside, Maqul, Aristotellachilensis, Cyanidin 3-,5-diglucoside, Matthiola incana, Four acylatedcyaniding 3-sambubloside-5 glucosides, Millet, Pernnisetum americanum,Cyanidin 3-glucoside, Cyanidin 3-glucoside, Mountain ash berry, Sorbusspp, Cyanidin 3-galactoside, Cyanidin 3,5-diglucoside Cyanidin 3-β-Dglucopyranoside, Mulberry, Morus nigra, Cyanidin 3-glucoside, Cyanidin3-glucoside, Cyanidin 3,5-diglucoside, Cyanidin 3-rutinoside, Cyanidin3-sophoroside, Myrtle berry, Myrtus communis, Cyanidin 3-glucosides,Cyanidin 3-glucosides, Cyanidin 3-diglucosides, Olive, Olea europaea,Cyanidin 3-rutinoside, Cyanidin 3-glucoside, Cyanidin 3-glucoside,Cyanidin derivatives, Onion, Allium sepa, Cyanidin 3-glucoside, Cyanidin3-glucoside, Cyanidin 3-diglucoside, Cyanidin 3-laminarioside, Orange,Citrus sinensis, Cyanidin 3-glucoside (95%), Cyanidin 3-glucoside,Cyanidin 3,5-diglucoside, Passion fruit, Pasiflora edulis, Cyanidin3-glucoside, Cyanidin 3-glucoside, Pea, Pisum sativurn, Cyanidin3-sophoroside glucosides, Cyanidin 3-sambubioside-5-glucosides, Peach,Prunus persica, Cyanidin 3-glucoside, Cyanidin 3-glucoside, Cyanidin3-rutinoside, Cyanidin derivatives, Peanut, Arachis hypogaea, Cyanidinglucosides, Pear, Pyrus communis, Cyanidin 3-galactoside, Cyanidin3-galactoside, Cyanidin 3-arabinoside, Cyanidin 3-arabinoside, Perilla,Perilla frutescens, Cyanidin 3,5-diglucoside, Cyanidin 3,5-derivatives,Petunia spp, Cyanidin 3-rutinoside, Phalsa, Grewia spp, Cyanidin3-glucoside, Cyanidin 3-glucoside, Pineapple, Anans comosus, Cyanidin3-galactoside, Cyanidin 3-galactoside, Pistachio, Pistacia vera,Pragmites australis, Cyanidin-3 derivatives, Plum, 2000 varieties, 15species, Cyanidin-glucoside (37%), Cyanidin glucoside,Cyanidin-rutinoside (45%), Pomegranate, Punica granatam,Cyanidin-glucoside (30%), Cyanidin-glucoside, Cyanidin-diglucoside(17%), Purple carrot, Daucus carota, Cyanidin-glucoside,Cyanidin-glucoside, Cyanidin-glucosylgalactoside, Cyanidin-galactoside,Cyanidin-digalactoside, Cyanidin-galactoside, Quince, Cydonia oblonga,Cyanidin-3 glucoside, Cyanidin 3,5-diglucoside, Cyanidin derivatives,Radish, Raphanus sativus, Acylated cyanidin 3-sophoroside-5-glucoside,Acylated cyanidin 3 diglucoside-5-glucoside, Red cabbage, Brassicaoleracea var capitata, Cyanidin glycosides, Reed, Phalaris arundinacea,Cyanidin 3-glucoside, Cyanidin 3-glucoside, Cyanidin3-(6″-,malonylglucoside), Cyanidin 3 (3″,6″dimalonyl-glucoside), Redonion, Allium cepa, Cyanidin 3-glucoside, Cyanidin 3-glucoside, Acylatedcyanidin 3-glucoside derivatives, Red petunia, Petunia spp, Cyanidin3-glucoside, Cyanidin 3-glucoside, Cyanidin 3-sophoroside, Redraspberry, Rubus idaeus, Cyanidin glucoside (17%), Cyanidin-glucoside,Cyanidin-rutinoside (7%), Cyanidin-sophoroside (50%), Cyanidinglycosylrutinoside (26%), Cyanidin-diglucoside, Rhubarb, Rneum spp,Cyanidin 3-glucoside, Cyanidin 3-glucoside, Cyanidin 3-rutinoside, Rice,Oryza spp, Cyanidin 3-glucoside, Cyanidin 3-glucoside, Cyanidin3-rhamnoside, Cyanidin 3,5-diglucoside, Rosehip, Rosa canina, Cyanidin3-rutinoside, Cyanidin 3-glucoside, Cyanidin 3-glucoside, Cyanidin3,5-diglucoside, Rye, Secale cereale, Cyanidin 3-glucoside, Cyanidin3-glucoside, Cyanidin 3-rhamnosylglucoside, Cyanidin3-rhamnosyldiglucoside, Cyanidin 3-rutinoside, Cyanidin 3-rutinosidederivatives, Cyanidin 3-gentiobioside, Sheepberry, Vibumum spp, Cyanidin3-glucoside, Cyanidin 3-glucoside, Cyanidin 3-arabinosylsambubioside,Sorghum, Sorghum bicolor, Cyanidin, Cyanidin glycosides, Sparkleberry,V. arboreum, Cyanidin 3-glucoside, Cyanidin 3-glucoside, Cyanidin3-arabinoside, Cyanidin 3-galactoside, Strawberry, Fragaria ananassa,Cyanidin-glucoside (minor), Cyanidin-glucoside, Sunflower, Hellanthusannuus, Cyanidin 3-glucoside, Cyanidin 3-glucoside, Acylated cyanidin3-glucoside, Cyanidin 3-xyloside, Cyanidin 3-xyloside, Acylated cyanidin3-xyloside, Cyanidin 3-vanillyl sambubioside, Sweet cherry, Prunusavintn, Cyanidin-glucoside, Cyanidin-glucoside, Cyanidin-rutinoside;Cyanidin 3-suphoroside, Sweet potato, Ipornoea batatas Sophronitiscoccinea, Cyanidin derivatives, Five acylated cyanidin3,3′,7-triglucosides, Tamarillo or tomato tree, Cyphomandrea betacea,Cyanidin 3-rutinoside, Cyanidin 3-glucoside, Cyanidin 3-glucoside,Tamarind, Tamarindus indica, Cyanidin 3-glucoside, Cyanidin 3-glucoside,Taro, Colocasia esculenta, Cyanidin 3-glucoside, Cyanidin 3-glucoside,Cyanidin 3-rutinoside, Tart Cherry (balaton), Prunus cerasus cv.Balaton, Nutrilite, Cyanidin-3-rutinoside-hexose (75%),Cyanidin-3-rutinoside-pentose (3%), Cyanidin-3-rutinoside (18%), Tartcherry (montmorency), Prunus cerasus cv. Montmorency, Nutrilite,Cyanidin-3-sophoroside (80%), Cyanidin-3-glucoside (20%),Cyanidin-3-glucoside (20%), Tulip, Tulipa spp, Cyanidin3-O-(6″-rhamnosylglucosides), Cyanidin 3-O-derivative, Turnip, brissicarapa, Cyanidin 3-glucoside, Cyanidin 3-glucoside, Cyanidin3-diglucoside-5-glucoside, Water lily, Nymphasa alba, Cyanidin3-O-(6″-acetyl-beta-galactopyrosinase (23%), Cyanidin 3-O-galactoside(2%), Cyanidin 3-O-galactoside (2%), Weigela spp, Cyanidin3-O-glucoside, Cyanidin 3-O-glucoside, Cyanidin 3-O-glucoside xylose,Wheat, Triticum spp, Cyanidin 3-glucoside, Cyanidin 3-glucoside,Acylated cyanidin glucoside, Cyanidin 3-rutinoside, Acylated cyanidin3-rutinoside, Cyanidin 3-gentiobioside, Wild rice, Zizania aquatica,Cyanidin 3-glucoside, Cyanidin 3-glucoside, Cyanidin 3-rhamnoglucoside,and Yam, Dioscoracea spp, Cyanidin 3,5-diglucoside, Cyanidin3-glucoside, Cyanidin 3-glucoside, Cyanidin 3-rhamnoglucoside, Cyanidin3-gentiobioside, Acylated cyanidin glucosides.

The term “anthocyanin” as used herein is intended to refer not only tomonomeric anthocyanins, but also refers to dimeric and polymeric (i.e.containing from 3 to 20 anthocyanidin monomer residues) forms ofanthocyanins and to leucoanthocyanidins (also known asflavan-3,4-diols). The anthocyanins can comprise substitutions (e.g.alkyl, alkoxy groups etc.) and in particular can be O-glycosylated, asdescribed above.

The anthocyanin in the composition can be a single anthocyanin, orcomprise a mixture of anthocyanins. In particular, the anthocyanin isselected from the group consisting of: malvidin, cyanidin, delphinidin,paeonidin, pelargonidin and petunidin, and glycosides thereof A typicalexample is malvin (malvidin diglucoside) chloride, which is commerciallyavailable in a purified form. Alternatively the anthocyanin can beobtained by extracting anthocyanin containing plants such as grape,black carrot, red cabbage, blackberry, blackcurrent, cranberry and thelike as described above.

The phrase “stabilized anthocyanin composition” as used herein isintended to mean that the anthocyanin, either as a glycoside(anthocyanoside) or as an aglycon (anthocyanidin), for example, at about37° C., pH=about 7.0, remains at least about 50% undegraded from theoriginal percentage of anthocyanin for at least about 3.5 hours.Likewise, the phrase includes pH values of about 4, about 5, about 6 andabout 8 with similar stability.

The term “stabilizing compound” as used herein is intended to includethose compounds that have at least one —SH group. Not to be limited bytheory, it is believed that an interaction occurs between the thiolgroup and the anthocyanin such that the thiol containing group isoxidized (often to a disulfide, —S—S—) and the anthocyanin receives ahydrogen atom, which is then later liberated.

Suitable examples of stabilizing compounds include yeast extract (e.g.,beer yeast), dihydrolipoic acid, salts of dihydrolipoic acid such asamino acid salts, cysteine, derivatives of cysteine, such asN-acetylcysteine, glutathione, salts of glutathione, SH-proteinase suchas papain, bromelain, ficin, ehymopapain and mixtures thereof,SH-metalloproteinases, peptides containing cysteine, peptides containingglutathioine, fermented oyster extract, fermented bean curd, thiolatedchitosans, thiolated gelatins or mixtures thereof.

In one aspect, the mole ratio of stabilizing compound to anthocyanin isbetween about 0.1 to about 10, more particularly between about 0.5 toabout 5 and even more particularly about 1 to about 1.

Not to be limited by theory the following is an explanation of thesurprising an unique findings with regard to the present invention.Anthocyanosides are susceptible to pH-shifts by forming colorlessderivatives. The flavylium-cation and the quinonoidal-base are coloredand the equilibria are not generally influenced with the presence ofboth species being pH-dependent [pH-value <2]. (See Scheme 1) It isbelieved that this equilibrium is established very quickly and that thereaction is reversible. The “attack” of the water under appropriateconditions yields a series of colorless derivatives (carbinolbase,chalcone). This part of the reaction progresses slowly is and notnormally reversible. The decolouration process begins at pH-values ofabout >3.5. Prevention of the water attack by molecular protectionprovides stabilization of anthocyanosides as described throughout thespecification.

Based on theory, known phenomenon of co-pigmentation (describingincreased stability of anthocyanosides in presence of “copigments”)yields a bathochromic (λ max shift towards higher wave length) and ahyperchromic (increase in absorption at λ max for the sameconcentrations) effect. Hence, clarification of whether thiol-compoundswere able to act as co-pigments was undertaken.

Based on theory, anthocyanosides are able to increase their stability byself-association. The higher the concentration, the more likely is theeffect of self-association. Self-association occurs at concentrations ofabout >0.0001 molar solution concentration. Self association may helpmask the protective effects of thiol-compounds; hence the protectiveeffects were tested at various concentrations.

Based on theory, co-pigmentation should yield a bathochromic (λ maxshift towards higher wave length) and a hyperchromic (increase inabsorption at λ max at the same concentration) shift.

The present studies based on UV-spectroscopy found that neither DHLA(dihydrolipoic acid) nor GSH (glutathione) yielded a bathochromic or ahyperchromic effect as shown in FIGS. 1 and 2. Therefore, increase instability is most likely related to another mechanism.

The spectral properties of freshly dissolved cyanidin-3-O-glucosides(protected and unprotected) in terms of λ max shifts for differentpH-values were identical as shown in FIGS. 1 and 2.

The stability of bilberry extract (various concentrations, molarconcentrations expressed as cyanidin-3-O-glucoside) was investigated inpresence/absence of GSH or DHLA by UV-spectroscopy measuring theabsorption at the pH-dependent λ max.

As noted in FIGS. 3, 4 and 5, the present investigations showed thatanthocyanosides are protected against degradation by GSH (0.65 mmolar)and DHLA (1.44 mmolar) at all concentrations tested, especially at pHvalues >5-6. The λ max at the various pH tested did not differ betweenthe unprotected and the protected solutions. Since it was observed thatwith cyanidin-glucoside no hyperchromic effect occurred, the stabilizingeffect of thiol-containing protection agents is considered to bedifferent from co-pigmentation.

The stability of bilberry extract at a 0.0001 molar concentration(expressed as cyanidin-3-O-glucoside) was investigated byUV-spectroscopy at different pH-values over time in absence/presence ofDHLA (1.44 mmolar solution).

These experiments shown in FIGS. 6A through 6D, demonstrate that DHLAprotects anthocyanosides at those pH-values where unprotectedanthocyanosides degrade quickly (pH>5-6). Most interestingly, whenfitting the data to statistical models (regression analysis) unprotecteddegradation kinetics fits to linear regression whereas DHLA protecteddegradation kinetics fits to exponential regression analysis. Thatpoints to a protection mechanism which builds up over time.

The degradation kinetics of selected anthocyanosides from bilberryextract (0.48 mmolar solution) was investigated over time by HPLC afterincubation at pH=7 and 25° C. in presence/absence of GSH (0.65 mmolarsolution).

At selected time points the peak area of Delphinidin-3-O-galactoside(very susceptible to degradation), Petunidin-3-O-galactoside (mediumsusceptibility) and Cyanidin-3-O-galactoside (rather stable) was checkedby HPLC (See FIGS. 7, 8 and 9). Stabilizing properties were observed inpresence of GSH, again better fitted by exponential regression analysis.It is therefore reasonable to conclude that the protection mechanism isdifferent from co-pigmentation and involves a mechanism where theprotective effect is built up over time.

Cyanidn-3-O-Galactoside (0.001 molar solution) was dissolved at pH=3.1or pH=7.0 each with/without DHLA (1.44 mmolar solution). Replicateincubations were followed at 25° C. over 3 days by HPLC.

As seen in FIG. 10, the effect of DHLA is surprisingly long-lasting.After 3 days at pH=7.0 at 25° C. about 47% of Cyanidn-3-O-Galactosidewas recovered in presence of DHLA, whereas the unprotected sampleyielded almost 0% of cyanidin-3-O-galactoside. Comparative figures forpH=3.1 were 95% and 61%, respectively.

The degradation kinetics of anthocyanosides from bilberry extract (0.48mmolar solution) was investigated in presence/absence of protecting DHLA(FIG. 11) (1.44 mmolar solution) and GSH (0.65 mmolar solution) by HPLC(FIG. 12).

The focus of the evaluation was whether the basic anthocyanidin skeletonis involved with the protection effect to some degree. When looking atthe results obtained, it seemed plausible that the protection effect ofthiol-compounds was inversely correlated with the susceptibility ofunprotected degradation. The more susceptible the anthocyanidin skeletonwas, the better was the protective effect of GSH or DHLA. A slightlybetter absolute effect for GSH was been observed.

Substitution patterns of anthocyanidins present in bilberry extract arenoted in the table which follows:

Anthocyanidin Substitution pattern (B-ring) Delphinidin 3′, 4′, 5′ OHPetunidin 3′-OCH3, 4′, 5′ OH Malvidin 3′, 5′-OCH3, 4′ OH Cyanidin 3′, 4′OH Peonidin 3′-OCH3, 4′ OH

As noted in the table above, there is evidence for a structure relatedeffect of (a) degradation and (b) for a protective mechanism. First, thenumber of substitutions about the anthocyanin plays a role, and, second,within the same number of substitutions the presence of methoxy-groupsinfluences the stability.

The most susceptible anthocyanidin is delphinidin, bearing 3hydroxyl-groups at the B-ring. The most stable is peonidin, bearing 1hydroxyl-group and 1 methoxy-group (superior to 2 hydroxyl-groups as incyaniding).

The proposed newly discovered mechanism is believe to be related to thestabilization of the flavylium cation at pH=7.0. All compounds tested dobear carboxylic acid groups and free thiol-groups.

Cysteine has two anchor points with anthocyanosides. First, there is astrong interaction of the thiol-portion (probably tightly associatedwith the anthocyanidin at position 4 or even to the sugar part) and,second, the carboxylic acid function protects the cationic part.

A similar mechanism is proposed for DHLA. The presence of thiol groupsis believed to anchor DHLA to the anthocyanidin so that the carboxylicfunction can help protect the molecule. Possibly, an increased effect ofDHLA compared to cysteine might be allocated to a more stable“association” of two thiol groups when compared to the single thiolfound in cysteine.

Similarly it is possible to draw the reaction with reduced glutathione.In this case, superior activity is obtained by two carboxylic acidfunctions instead of one.

The mechanism is considered to be related to a reaction cascade of theflavylium cation while serving as radical scavenger.

The redox potential of 2 R—SH/R—SS—R compounds tested is lower than thatof anthocyanosides. For example 2 GSH/GSSG=˜−0.22 V whereas the redoxpotential A-OH/A=O=˜0.20-0.75 V (see below).

Radical scavenging properties of anthocyanosides can explained byfollowing reactions:

R=H Anthocyanidin, R=sugar Anthocyanoside

Quinone-/Semiquinone structure (colored products, examples).

The stability of these (semi)quinines is believed to be related to thesubstitution pattern, as the quinone structure requires a4′-substitution. Once the quinone is formed, reversal of the reaction tothe flavylium structure is less feasible. On the other hand, anysubstitution on 5′-position hinders the formation of the semiquinone.

That may explain why a 3′-OH and 4′-methoxy substituted structure appearto be most stable: It forms a semiquinone that cannot be attacked bywater forming colorless products. Such a semiquinone may be more or lesseasily re-converted to the flavylium cation.

Anthocyanidins/Anthocyanosides (A-OH) are converted tosemiquinones/quinones (A=O) by following reaction:A-OH->A=O+H⁺ +e ⁻

R—SH compounds undergo following reactions:

Overall process: 2 RSH -> R—SS—R + 2 H⁺+ 2 e⁻ Detailed: R—SH -> R—S— +H⁺-> R-S° + e⁻+ H⁺ 2 R—S° -> R—SS—R (R-S° . . . Radical)

Such a mechanism would point to a 1:1 consumption in a way that 2molecules anthocyanosides are able to form 1 molecule R—SS—R from 2molecules R—SH and are left as semiquinones. Alternatively 1anthocyanoside molecule may form 1R—SS—R by being converted to thequinone.

If no alternative reactions are taking place, following reaction cascadeis reasonable:

In a combination of R—SH and anthocyanosides at neutral pH, primarilyR—SH is converted to R—S°.

Part of the R—S° is scavenged by anthocyanosides (A-OH) which arethereby converted to A=O semi-quinones, yielding again R—SH.

At pH=7, A=O is reconverted to A-OH by consumption of R—SH and will theneventually degrade gradually as known.

Suitable examples of stabilizing compounds include yeast extract,dihydrolipoic acid, salts of dihydrolipoic acid such as amino acidsalts, cysteine, derivatives of cysteine, such as N-acetylcysteine,glutathione, salts of glutathione, SH-proteinase such as papain,bromelain, ficin, ehymopapain and mixtures thereof,SH-metalloproteinases, peptides containing cysteine, peptides containingglutathioine, fermented oyster extract, fermented bean curd, thiolatedchitosans, thiolated gelatins or mixtures thereof.

In one aspect, the mole ratio of stabilizing compound to anthocyaninextract is between about 0.1 to about 10, more particularly betweenabout 0.5 to about 5 and even more particularly about 1 to about 1.

In another aspect, the stabilized anthocyanin extract composition isstabile toward degradation when exposed to an aqueous environment with apH of about 2, about 3, about 4, about 5, etc. through about 14, forexample, a pH of 7 or greater.

In still another aspect, the stabilized anthocyanin extract is ananthocyanoside.

In still yet another aspect, the stabilized anthocyanin extract is ananthocyanidin.

In still other aspects of the invention, the stabilized anthocyaninextract includes one or more anthocyanosides that are glycosides ofperlargonidin, peonidin, cyanidin, malvidin, petunidin, delphinidin.

The present invention also pertains to methods of preparing thestabilized anthocyanin compositions described herein.

The present invention provides that the stabilizing compound(s) can beadmixed with the anthocyanin containing plant material during theextraction and/or manufacturing process, thereby reducing or eliminatingthe oxidative destruction of the anthocyanin that commonly occurs uponprocessing and even upon storage. For example one or more of thestabilizing compounds in the ratios generally described herein can beadded into the extraction medium (solvent) during the extraction processas disclosed in US Patent Publication 2002/0018821, published Feb. 14,2002 by Chrystele Soulier et al., the contents of which are incorporatedherein in their entirety.

Typically, the anthocyanin extract is mixed directly with thestabilizing compound. This can be accomplished by simply mixing,grinding, combining, etc. the two materials as solids or by dissolutionin a solvent, such as water. Additional additives, such as carriers,vitamins, antioxidants, etc., as described herein below, can be added tothe mixture by conventional methods.

In one embodiment, a red fruit extract containing anthocyanosides, istaken up in an aqueous solution and optionally treated with an —SHstabilizing compound as described herein. The aqueous extract is cooleduntil it reaches a homogeneous temperature of less than 15° C. Theaqueous extract is filtered and is optionally treated with an —SHstabilizing compound as described herein, the permeate obtained isrecovered and loaded onto a macrocrosslinked polymeric resin. The resinis then rinsed with demineralized water, optionally treated with an —SHstabilizing compound as described herein and then the resin obtained iseluted with an alcoholic eluting solution, which may optionally betreated with an —SH stabilizing compound as described herein. The eluateobtained is concentrated, optionally treated with an —SH stabilizingcompound as described herein and then dried.

In another embodiment, the process of stabilization is carried out on analcoholic red fruit extract obtained according to the following process.The pulp is first separated from the whole red fruit and the pulp isthen brought into contact with an alcoholic extraction solution whichcan optionally contain an —SH stabilizing compound as described herein.The solid phase is separated from the liquid phase and the liquid phasecan be optionally treated with an —SH stabilizing compound as describedherein. The major portion of the residual alcohol contained in theliquid phase is evaporated under vacuum so as to obtain an alcoholicconcentrate.

Advantageously, the solvent used for the alcoholic extraction is chosenfrom the group comprising methanol, ethanol, butanol and acetone.

In practice, the alcoholic extraction is carried out at room temperaturein at least two successive steps, each lasting 20 minutes. The solventis then evaporated off. In addition, it is also possible to carry outthe extraction of the anthocyanosides not from the pulp alone, but fromwhole fruits.

According to the invention, the process of stabilization can be carriedout starting with extracts of fruits which are commercially available orwith prepurified anthocyanoside extract, each provided in liquid orpowdered form. In this case, the fruit extract or the prepurifiedextract can then be taken up, before the purification step, either withalcohol, in particular methanol, or with water and treated with an —SHstabilizing compound as described herein.

In the process of purification of the invention, the cooling of the redfruit extract is advantageously carried out until the temperature of theextract is homogeneous and less than 10° C., in particular, less than 5°C., with the temperature being maintained for at least about twelvehours.

With regard to the step of filtration of the aqueous extract oralcoholic extract, it may be carried out on a cellulose filter or astainless steel gauze with a cut-off of between 0 and 100 micrometers orequivalent.

In order to further increase the titer and the concentration ofstabilized anthocyanosides in the final extract, the alcoholic solutionwith which the stabilized anthocyanosides are eluted from the resin isan aqueous solution of ethanol whose ethanol concentration is between 10and 90%, advantageously close to 40%.

The stabilized eluate obtained is concentrated at a controlledtemperature in the region of 30° C. and then freeze-dried or spray-driedso as to obtain a stabilized powder.

In one embodiment, for example, a concentrate of bilberry extract andL-cysteine hydrochloride are combined (9:1, w/w) and spray dried toafford a stabilized bilberry extract as a powder. In general, thebilberry extract to free cysteine ratio is approximately 10:1, w/w.

The present invention further pertains to methods of treatment ofvarious ailments by administration of a therapeutically effective amountof the stabilized anthocyanin compositions described herein. Ailmentsinclude, the need for increased antioxidant capacity, arthrosclerosis,reduction of pain, inflammation. Reduction or the elimination of painincludes various forms of pain including arthritis, dysmenorrheal,headaches, joint pain, muscular pain, osteoarthritis, age-relatedmacular degeneration (AMD), cataracts, retinopathy, and combinationsthereof.

Therefore, the present invention further provides bioavailablestabilized anthocyanin compositions that are useful to treat variousafflictions noted herein. The stabilized anthocyanin compositions can beadministered by a number of methods, as discussed infra.

Surprisingly, the present invention provides that use of cysteine incombination with an anthocyanin composition (whether it be ananthocyanidine or an anthocyanoside) helps to increase the delivery ofthe anthocyanin to a subject in need thereof by at least twice theamount relative to a subject that ingests an anthocyanin compositionwithout the presence of cysteine. It has been surprisingly found thatplasma concentration levels of the anthocyanin where the anthocyanin isdelivered in the presence of cysteine after 4 hours is at least twicethe plasma concentration of an anthocyanin delivered without thecysteine. Therefore, the present invention provides a method to increasethe amount of bioavailable anthocyanin in a subject by administering tothe subject an effective amount of an anthocyanin and cysteine. Theadministration can be by any means, but oral delivery is generallypreferred.

In one embodiment, the ratio of the anthocyanin to the cysteine is about10 to about 0.1, more particularly between about 10 to about 0.5, andeven more particularly between about 10 to about 1 on a weight basis. Itis important to note that without the use of cysteine, thebioavailability of the anthocyanin is dramatically decreased as noted inthe examples described herein.

The increased bioavailability an anthocyanin in the presence of cysteineis generally at least twice that the bioavailability of an anthocyaninalone after administration. Ideally, the bioavailability is increased bythe inclusion of cysteine such that the bioavailability increases to 3,4, 5, 10 and 20 times more than equivalent anthocyanins without theinclusion of cysteine. This is a surprising find.

The compositions of the invention can be incorporated into variousfoods, drinks, snacks, etc. In one aspect, the composition can besprinkled onto a food product, prior to consumption. If sprinkled onto afood product, a suitable carrier such as starch, sucrose or lactose, canbe used to help distribute the concentration of the stabilizedanthocyanin composition making it easier to apply to the food product.

The compositions of the present invention can also be provided assupplements in various prepared food products. For the purposes of thisapplication, prepared food product means any natural, processed, diet ornon-diet food product to which a composition of the invention has beenadded. The compositions of the present invention can be directlyincorporated into many prepared diet food products, including, but notlimited to diet drinks, diet bars and prepared frozen meals.Furthermore, the compositions of the inventions can be incorporated intomany prepared non-diet products, including, but not limited to candy,snack products such as chips, prepared meat products, milk, cheese,yoghurt, sport bars, sport drinks, mayonnaise, salad dressing, bread andany other fat or oil containing foods. As used herein, the term “foodproduct” refers to any substance fit for human or animal consumption.

The compositions of the invention can be added to various drinks, suchas fruit juices, milkshakes, milk, etc.

The preferred method of administration is oral. The compositions of theinvention can be formulated with suitable carriers such as starch,sucrose or lactose in tablets, capsules, solutions, syrups andemulsions. The tablet or capsule of the present invention can be coatedwith an enteric coating that dissolves at a pH of about 6.0 to 7.0. Asuitable enteric coating, which dissolves in the small intestine but notin the stomach, is cellulose acetate phthalate.

Formulation of the compositions of the invention into a soft gel capsulecan be accomplished by many methods known in the art. Often theformulation will include an acceptable carrier, such as an oil, or othersuspending or emulsifying agent.

Suitable optional carriers include but are not limited to, for example,fatty acids, esters and salts thereof, that can be derived from anysource, including, without limitation, natural or synthetic oils, fats,waxes or combinations thereof. Moreover, the fatty acids can be derived,without limitation, from non-hydrogenated oils, partially hydrogenatedoils, fully hydrogenated oils or combinations thereof. Non-limitingexemplary sources of fatty acids (their esters and salts) include seedoil, fish or marine oil, canola oil, vegetable oil, safflower oil,sunflower oil, nasturtium seed oil, mustard seed oil, olive oil, sesameoil, soybean oil, corn oil, peanut oil, cottonseed oil, rice bran oil,babassu nut oil, palm oil, low erucic rapeseed oil, palm kernel oil,lupin oil, coconut oil, flaxseed oil, evening primrose oil, jojoba,wheat germ oil, tallow, beef tallow, butter, chicken fat, lard, dairybutterfat, shea butter or combinations thereof.

Specific non-limiting exemplary fish or marine oil sources includeshellfish oil, tuna oil, mackerel oil, salmon oil, menhaden, anchovy,herring, trout, sardines or combinations thereof. In particular, thesource of the fatty acids is fish or marine oil (DHA or EPA), soybeanoil or flaxseed oil. Alternatively or in combination with one of theabove identified carrier, beeswax can be used as a suitable carrier, aswell as suspending agents such as silica (silicon dioxide).

The formulations of the invention are also considered to benutraceuticals. The term “nutraceutical” is recognized in the art and isintended to describe specific chemical compounds found in foods that canprevent disease or ameliorate an undesirable condition.

The formulations of the invention can further include variousingredients to help stabilize, or help promote the bioavailability ofthe components of the beneficial compositions of the invention or serveas additional nutrients to an individual's diet. Suitable additives caninclude vitamins and biologically-acceptable minerals. Non-limitingexamples of vitamins include vitamin A, B vitamins, vitamin C, vitaminD, vitamin E, vitamin K and folic acid. Non-limiting examples ofminerals include iron, calcium, magnesium, potassium, copper, chromium,zinc, molybdenum, iodine, boron, selenium, manganese, derivativesthereof or combinations thereof. These vitamins and minerals can be fromany source or combination of sources, without limitation. Non-limitingexemplary B vitamins include, without limitation, thiamine, niacinamide,pyridoxine, riboflavin, cyanocobalamin, biotin, pantothenic acid orcombinations thereof.

Various additives can be incorporated into the present compositions.Optional additives of the present composition include, withoutlimitation, hyaluronic acid, phospholipids, starches, sugars, fats,antioxidants, amino acids, proteins, flavorings, coloring agents,hydrolyzed starch(es) and derivatives thereof or combinations thereof.

As used herein, the term “antioxidant” is recognized in the art andrefers to synthetic or natural substances that prevent or delay theoxidative deterioration of a compound. Exemplary antioxidants includetocopherols, flavonoids, catechins, superoxide dismutase, lecithin,gamma oryzanol; vitamins, such as vitamins A, C (ascorbic acid) and Eand beta-carotene; natural components such as camosol, camosic acid androsmanol found in rosemary and hawthorn extract, proanthocyanidins suchas those found in grapeseed or pine bark extract, and green tea extract.

Compositions comprising the stabilized anthocyanin compositions of theinvention can be manufactured by methods of conventional mixing,dissolving, granulating, dragee-making levigating, emulsifying,encapsulating, entrapping or lyophilization processes. The compositionscan be formulated in conventional manner using one or morephysiologically acceptable carriers, diluents, excipients or auxiliariesthat facilitate processing of the stabilized anthocyanin compositionsinto preparations that can be used.

The compositions of the invention can take a form suitable for virtuallyany mode of administration, including, for example, oral, buccal,systemic, injection, transdermal, rectal, vaginal, etc., or a formsuitable for administration by inhalation or insufflation.

Systemic formulations include those designed for administration byinjection, e.g., subcutaneous, intravenous, intramuscular, intrathecalor intraperitoneal injection, as well as those designed for transdermal,transmucosal oral or pulmonary administration.

Useful injectable preparations include sterile suspensions, solutions oremulsions of the stabilized anthocyanin compositions in aqueous or oilyvehicles. The compositions can also contain formulating agents, such assuspending, stabilizing and/or dispersing agent. The formulations forinjection can be presented in unit dosage form, e.g., in ampoules or inmultidose containers, and can contain added preservatives.

Alternatively, the injectable formulation can be provided in powder formfor reconstitution with a suitable vehicle, including but not limited tosterile pyrogen free water, buffer, dextrose solution, etc., before use.To this end, the stabilized anthocyanin compositions can be dried by anyart-known technique, such as lyophilization, and reconstituted prior touse.

For transmucosal administration, penetrants appropriate to the barrierto be permeated are used in the formulation. Such penetrants are knownin the art.

For oral administration, the compositions of the invention can take theform of, for example, lozenges, tablets or capsules prepared byconventional means with pharmaceutically acceptable excipients such asbinding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidoneor hydroxypropyl methylcellulose); fillers (e.g., lactose,microcrystalline cellulose or calcium hydrogen phosphate); lubricants(e.g., magnesium stearate, talc or silica); disintegrants (e.g., potatostarch or sodium starch glycolate); or wetting agents (e.g., sodiumlauryl sulfate). The tablets can be coated by methods well known in theart with, for example, sugars, films or enteric coatings.

Liquid preparations for oral administration can take the form of, forexample, elixirs, solutions, syrups or suspensions, or they can bepresented as a dry product for constitution with water or other suitablevehicle before use. Such liquid preparations can be prepared byconventional means with pharmaceutically acceptable additives such assuspending agents (e.g., sorbitol syrup, cellulose derivatives orhydrogenated edible fats); emulsifying agents (e.g., lecithin oracacia); non aqueous vehicles (e.g., almond oil, oily esters, ethylalcohol, or fractionated vegetable oils); and preservatives (e.g.,methyl or propyl p hydroxybenzoates or sorbic acid). The preparationscan also contain buffer salts, preservatives, flavoring, coloring andsweetening agents as appropriate.

Preparations for oral administration can be suitably formulated to givecontrolled release of the stabilized anthocyanin composition as is wellknown.

For buccal administration, the compositions can take the form of tabletsor lozenges formulated in conventional manner.

For rectal and vaginal routes of administration, the stabilizedanthocyanin compositions can be formulated as solutions (for retentionenemas) suppositories or ointments containing conventional suppositorybases such as cocoa butter or other glycerides.

For nasal administration or administration by inhalation orinsufflation, the stabilized anthocyanin compositions can beconveniently delivered in the form of an aerosol spray from pressurizedpacks or a nebulizer with the use of a suitable propellant, e.g.,dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, fluorocarbons, carbon dioxide or othersuitable gas. In the case of a pressurized aerosol, the dosage unit canbe determined by providing a valve to deliver a metered amount. Capsulesand cartridges for use in an inhaler or insufflator (for examplecapsules and cartridges comprised of gelatin) can be formulatedcontaining a powder mix of the compound and a suitable powder base suchas lactose or starch.

For prolonged delivery, the stabilized anthocyanin compositions can beformulated as a depot preparation for administration by implantation orintramuscular injection. The stabilized anthocyanin compositions can beformulated with suitable polymeric or hydrophobic materials (e.g., as anemulsion in an acceptable oil) or ion exchange resins, or as sparinglysoluble derivatives, e.g., as a sparingly soluble salt. Alternatively,transdermal delivery systems manufactured as an adhesive disc or patch,which slowly releases the stabilized anthocyanin compositions forpercutaneous absorption, can be used. To this end, permeation enhancerscan be used to facilitate transdermal penetration of the stabilizedanthocyanin compositions. Suitable transdermal patches are described infor example, U.S. Pat. No. 5,407,713; U.S. Pat. No. 5,352,456; U.S. Pat.No. 5,332,213; U.S. Pat. No. 5,336,168; U.S. Pat. No. 5,290,561; U.S.Pat. No. 5,254,346; U.S. Pat. No. 5,164,189; U.S. Pat. No. 5,163,899;U.S. Pat. No. 5,088,977; U.S. Pat. No. 5,087,240; U.S. Pat. No.5,008,110; and U.S. Pat. No. 4,921,475.

Alternatively, other delivery systems can be employed. Liposomes andemulsions are well-known examples of delivery vehicles that can be usedto deliver stabilized anthocyanin compositions. Certain organic solventssuch as dimethylsulfoxide (DMSO) can also be employed, although usuallyat the cost of greater toxicity.

The compositions can, if desired, be presented in a pack or dispenserdevice, which can contain one or more unit dosage forms containing thestabilized anthocyanin compositions. The pack can, for example, comprisemetal or plastic foil, such as a blister pack. The pack or dispenserdevice can be accompanied by instructions for administration.

Soft gel or soft gelatin capsules can be prepared, for example, withoutlimitation, by dispersing the formulation in an appropriate vehicle(e.g., rice bran oil, and/or beeswax) to form a high viscosity mixture.This mixture is then encapsulated with a gelatin based film usingtechnology and machinery known to those in the soft gel industry. Thecapsules so formed are then dried to constant weight. Typically, theweight of the capsule is between about 100 to about 2500 milligrams andin particular weigh between about 1500 and about 1900 milligrams, andmore specifically can weigh between about 1500 and about 2000milligrams.

For example, when preparing soft gelatin shells, the shell can includebetween about 20 to 70 percent gelatin, generally a plasticizer andabout 5 to about 60% by weight sorbitol. The filling of the soft gelatincapsule is liquid (principally a carrier such as rice bran oil or wheatgerm oil and/or beeswax if desired) and can include, apart from thestabilized anthocyanin compositions, a hydrophilic matrix. Thehydrophilic matrix, if present, is a polyethylene glycol having anaverage molecular weight of from about 200 to 1000. Further ingredientsare optionally thickening agents and/or emulsifying agent(s). In oneembodiment, the hydrophilic matrix includes polyethylene glycol havingan average molecular weight of from about 200 to 1000, 5 to 15%glycerol, and 5 to 15% by weight of water. The polyethylene glycol canalso be mixed with propylene glycol and/or propylene carbonate.

In another embodiment, the soft gel capsule is prepared from gelatin,glycerine, water and various additives. Typically, the percentage (byweight) of the gelatin is between about 30 and about 50 weight percent,in particular between about 35 and about weight percent and morespecifically about 42 weight percent. The formulation includes betweenabout 15 and about 25 weight percent glycerine, more particularlybetween about 17 and about 23 weight percent and more specificallyabout, 20 weight percent glycerine.

The remaining portion of the capsule is typically water. The amountvaries from between about 25 weigh percent and about 40 weight percent,more particularly between about 30 and about 35 weight percent, and morespecifically about 35 weight percent. The remainder of the capsule canvary, generally, between about 2 and about 10 weight percent composed ofa flavoring agent(s), sugar, coloring agent(s), etc. or combinationthereof. After the capsule is processed, the water content of the finalcapsule is often between about 5 and about 10 weight percent, moreparticularly 7 and about 12 weight percent, and more specificallybetween about 9 and about 10 weight percent.

As for the manufacturing, it is contemplated that standard soft shellgelatin capsule manufacturing techniques can be used to prepare thesoft-shell product. Examples of useful manufacturing techniques are theplate process, the rotary die process pioneered by R. P. Scherer, theprocess using the Norton capsule machine, and the Accogel machine andprocess developed by Lederle. Each of these processes are maturetechnologies and are all widely available to any one wishing to preparesoft gelatin capsules.

Emulsifying agents can be used to help solubilize the ingredients withinthe soft gelatin capsule. Specific examples of the surfactant,emulsifier, or effervescent agent include D-sorbitol, ethanol,carrageenan, carboxyvinyl polymer, carmellose sodium, guar gum,glycerol, glycerol fatty acid ester, cholesterol, white beeswax, dioctylsodium sulfosuccinate, sucrose fatty acid ester, stearyl alcohol,stearic acid, polyoxyl 40 stearate, sorbitan sesquioleate, cetanol,gelatin, sorbitan fatty acid ester, talc, sorbitan trioleate, paraffin,potato starch, hydroxypropyl cellulose, propylene glycol, propyleneglycol fatty acid ester, pectin, polyoxyethylene (105) polyoxypropylene(5) glycol, polyoxyethylene (160) polyoxypropylene (30) glycol,polyoxyethylene hydrogenated castor oil, polyoxyethylene hydrogenatedcastor oil 40, polyoxyethylene hydrogenated castor oil 60, polyoxyl 35castor oil, polysorbate 20, polysorbate 60, polysorbate 80, macrogol400, octyldodecyl myristate, methyl cellulose, sorbitan monooleate,glycerol monostearate, sorbitan monopalmitate, sorbitan monolaurate,lauryl dimethylamine oxide solution, sodium lauryl sulfate,lauromacrogol, dry sodium carbonate, tartaric acid, sodium hydroxide,purified soybean lecithin, soybean lecithin, potassium carbonate, sodiumhydrogen carbonate, medium-chain triglyceride, citric anhydride, cottonseed oil-soybean oil mixture, and liquid paraffin.

The present invention also provides packaged formulations of thecompositions of the invention and instructions for use of the productfor appropriate condition(s). Typically, the packaged formulation, inwhatever form, is administered to an individual in need thereof.Typically, the dosage requirement is between about 1 to about 4 dosagesa day.

Although the present invention describes the preparation, use,manufacture and packaging of the compositions of the invention in softgelatin capsules for treatment of various conditions, it should not beconsidered limited to only soft gelatin capsules. Ingestiblecompositions of the invention can be delivered in traditional tablets,pills, lozenges, elixirs, emulsions, hard capsules, liquids,suspensions, etc. as described above.

The stabilized anthocyanin compositions of the invention, orcompositions thereof, will generally be used in an amount effective toachieve the intended result, for example in an amount effective to treator prevent the particular inflammatory related condition being treated.The composition can be administered therapeutically to achievetherapeutic benefit or prophylactically to achieve prophylactic benefit.By therapeutic benefit is meant eradication or amelioration of theunderlying disorder being treated and/or eradication or amelioration ofone or more of the symptoms associated with the underlying disorder suchthat the patient reports an improvement in feeling or condition,notwithstanding that the patient can still be afflicted with theunderlying disorder. For example, administration of a composition of theinvention to a patient suffering from pain provides therapeutic benefitnot only when the underlying condition is eradicated or ameliorated, butalso when the patient reports a decrease in the severity or duration ofthe physical discomfort associated with the pain.

For prophylactic administration, the composition can be administered toa patient at risk of developing one of the previously describedconditions.

The amount of composition administered will depend upon a variety offactors, including, for example, the particular indication beingtreated, the mode of administration, whether the desired benefit isprophylactic or therapeutic, the severity of the indication beingtreated and the age and weight of the patient, etc. Determination of aneffective dosage is well within the capabilities of those skilled in theart.

Total dosage amounts of a stabilized anthocyanin composition willtypically be in the range of from about 0.0001 or 0.001 or 0.01mg/kg/day to about 100 mg/kg/day, but may be higher or lower, dependingupon, among other factors, the activity of the components, itsbioavailability, the mode of administration and various factorsdiscussed above. Dosage amount and interval can be adjusted individuallyto provide plasma levels of the compound(s) which are sufficient tomaintain therapeutic or prophylactic effect. For example, the compoundscan be administered once per week, several times per week (e.g., everyother day), once per day or multiple times per day, depending upon,among other things, the mode of administration, the specific indicationbeing treated and the judgment of the prescribing physician. Skilledartisans will be able to optimize effective local dosages without undueexperimentation.

The following paragraphs enumerated consecutively from 1 through 91provide for various aspects of the present invention. In one embodiment,in a first paragraph (1), the present invention provides a stabilizedanthocyanin extract composition comprising an anthocyanin extract and astabilizing compound having at least one —SH group with the provisiothat the stabilizing compound is not glutathione.

2. The stabilized anthocyanin extract composition of paragraph 1,wherein the stabilizing compound is yeast extract, dihydrolipoic acid,derivatives of dihydrolipoic acid, cysteine, derivatives of cysteine,SH-proteinase, SH-metalloproteinase, peptides containing cysteine,peptides containing glutathione, fermented oyster extract, thiolatedchitosan, thiolated gelatin or mixtures thereof.

3. The stabilized anthocyanin extract composition of either paragraphs 1or 2, wherein the anthocyanin extract is bilberry extract, blackcurrantextract, cranberry extract, black soybean extract, cowberry extract,blueberry extract or mixtures of two or more thereof.

4. The stabilized anthocyanin extract composition of any of paragraphs 1through 3, wherein the mole ratio of stabilizing compound to anthocyaninextract is about 0.1 to about 10.

5. The stabilized anthocyanin extract composition of any of paragraphs 1through 4, wherein the composition is stabile toward degradation whenexposed to an aqueous environment with a pH of between about 2 and about12.

6. The stabilized anthocyanin extract composition of any of paragraphs 1through 5, wherein the anthocyanin extract comprises an anthocyanoside.

7. The stabilized anthocyanin extract of paragraph 1, wherein theanthocyanin extract includes an anthocyanoside.

8. The stabilized anthocyanin extract of paragraph 7, wherein theanthocyanoside is a glycoside of perlargonidin, peonidin, cyanidin,malvidin, petunidin, delphinidin or mixtures of two or more thereof.

9. A stabilized anthocyanin extract composition comprising ananthocyanin extract and a stabilizing compound having at least one —SHgroup.

10. The stabilized anthocyanin extract composition of paragraph 9,wherein the stabilizing compound is yeast extract, dihydrolipoic acid,derivatives of dihydrolipoic acid, cysteine, derivatives of cysteine,glutathione, derivatives of glutathione, SH-proteinase,SH-metalloproteinase, peptides containing cysteine, peptides containingglutathione, fermented oyster extract, thiolated chitosan, thiolatedgelatin or mixtures thereof.

11. The stabilized anthocyanin extract composition of either ofparagraphs 9 or 10, wherein the anthocyanin extract is bilberry extract,blackcurrant extract, cranberry extract, black soybean extract, cowberryextract, blueberry extract or mixtures of two or more thereof.

12. The stabilized anthocyanin extract composition of any of paragraphs9 through 11, wherein the mole ratio of stabilizing compound toanthocyanin extract is about 0.1 to about 10.

13. The stabilized anthocyanin extract composition of any of paragraphs9 through 12, wherein the composition is stabile toward degradation whenexposed to an aqueous environment with a pH of between about 2 and about12.

14. The stabilized anthocyanin extract of any of paragraphs 9 through13, wherein the anthocyanin extract comprises an anthocyanoside.

15. The stabilized anthocyanin extract of paragraph 9, wherein theanthocyanin extract includes an anthocyanoside.

16. The stabilized anthocyanin extract of paragraph 15, wherein theanthocyanoside is a glycoside of perlargonidin, peonidin, cyanidin,malvidin, petunidin, delphinidin or mixtures of two or more thereof.

17. A method to stabilize an anthocyanin extract composition comprisingthe step of combining an anthocyanin extract with a sufficient amount ofa stabilizing compound having at least one —SH group with the provisothat the stabilizing compound is not reduced glutathione, such that theanthocyanin extract is stabilized.

18. The method of paragraph 17, wherein the stabilizing compound isyeast extract, dihydrolipoic acid, derivatives of dihydrolipoic acid,cysteine, derivatives of cysteine, SH-proteinase, SH-metalloproteinase,peptides containing cysteine, peptides containing glutathioine,fermented oyster extract, thiolated chitosan, thiolated gelatin ormixtures thereof.

19. The method of either of paragraphs 17 or 18, wherein the anthocyaninextract is bilberry extract, blackcurrant extract, cranberry extract,black soybean extract, cowberry extract, blueberry extract or mixturesof two or more thereof.

20. The method of any of paragraphs 17 through 19, wherein the moleratio of stabilizing compound to anthocyanin extract is about 0.1 toabout 10.

21. The method of any of paragraphs 17 through 20, wherein thecomposition is stabile toward degradation when exposed to an aqueousenvironment with a pH of between about 2 and about 12.

22. The method of any of paragraphs 17 through 21, wherein theanthocyanin extract comprises an anthocyanoside.

23. The method of paragraph 17, wherein the anthocyanin extract includesan anthocyanoside.

24. The method of paragraph 23, wherein the anthocyanoside is aglycoside of perlargonidin, peonidin, cyanidin, malvidin, petunidin,delphinidin or mixtures of two or more thereof.

25. The method of paragraph 17, wherein the stabilized anthocyaninextract composition is stabile at ambient conditions for at least aboutone day.

26. The method of paragraph 22, wherein the stabilized anthocyanoside isstable at a pH of 7 for at least about 6 hours.

27. The method of paragraph 26, wherein the stabilized anthocyanosidemaintained at a pH of 7 retains at least 50% of the anthocyanin as aglycoside over about 4 hours.

28. A method to stabilize an anthocyanin extract composition comprisingthe step of combining an anthocyanin extract with a sufficient amount ofa stabilizing compound having at least one —SH group, such that theanthocyanin is stabilized.

29. The method of paragraph 28, wherein the stabilizing compound isyeast extract, dihydrolipoic acid, derivatives of dihydrolipoic acid,cysteine, derivatives of cysteine, glutathione, derivatives ofglutathione, SH-proteinase, SH-metalloproteinase, peptides containingcysteine, peptides containing glutathioine, fermented oyster extract,thiolated chitosan, thiolated gelatin or mixtures thereof.

30. The method of either of paragraphs 28 or 29, wherein the anthocyaninextract is bilberry extract, blackcurrant extract, cranberry extract,black soybean extract, cowberry extract, blueberry extract or mixturesof two or more thereof.

31. The method of any of paragraphs 28 through 30, wherein the moleratio of stabilizing compound to anthocyanin extract is about 0.1 toabout 10.

32. The method of any of paragraphs 28 through 31, wherein thecomposition is stabile toward degradation when exposed to an aqueousenvironment with pH of between about 2 and about 12.

33. The method of any of paragraphs 28 through 32, wherein theanthocyanin extract comprises an anthocyanoside.

34. The method of paragraph 28, wherein the anthocyanin extract includesan anthocyanoside.

35. The method of paragraph 34, wherein the anthocyanoside is aglycoside of perlargonidin, peonidin, cyanidin, malvidin, petunidin,delphinidin or mixtures of two or more thereof.

36. The method of paragraph 28, wherein the stabilized anthocyaninextract composition is stabile at ambient conditions for at least aboutone day.

37. The method of paragraph 33, wherein the stabilized anthocyanoside isstable at a pH of 7 for at least about six hours.

38. The method of paragraph 37, wherein the stabilized anthocyanosidemaintained at a pH of 7 retains at least 50% of the anthocyanin as aglycoside over about 4 hours.

39. A pH stabilized anthocyanin extract composition comprising ananthocyanin extract and a stabilizing compound having at least one —SHgroup.

40. The pH stabilized anthocyanin extract composition of paragraph 39,wherein the stabilizing compound is yeast extract, dihydrolipoic acid,derivatives of dihydrolipoic acid, cysteine, derivatives of cysteine,glutathione, derivatives of glutathione, SH-proteinase,SH-metalloproteinase, peptides containing cysteine, peptides containingglutathioine, fermented oyster extract, thiolated chitosan, thiolatedgelatin or mixtures thereof.

41. The pH stabilized anthocyanin extract composition of either ofparagraphs 39 or 40, wherein the anthocyanin extract is bilberryextract, blackcurrant extract, cranberry extract, black soybean extract,cowberry extract, blueberry extract or mixtures of two or more thereof.

42. The pH stabilized anthocyanin extract composition of any ofparagraphs 39 through 41, wherein the mole ratio of stabilizing compoundto anthocyanin extract is about 0.1 to about 10.

43. The pH stabilized anthocyanin extract composition of any ofparagraphs 39 through 42, wherein the composition is stabile towarddegradation when exposed to an aqueous environment with a pH of betweenabout 2 and about 12.

44. The pH stabilized anthocyanin extract composition of any ofparagraphs 39 through 43, wherein the anthocyanin extract comprises ananthocyanoside.

45. The pH stabilized anthocyanin extract composition of paragraph 39,wherein the anthocyanin extract includes an anthocyanoside.

46. The pH stabilized anthocyanin extract composition of paragraph 45,wherein the anthocyanoside is a glycoside of perlargonidin, peonidin,cyanidin, malvidin, petunidin, delphinidin or mixtures of two or morethereof.

47. The pH stabilized anthocyanin extract composition of paragraph 39,wherein the stabilized anthocyanin extract is stabile at ambientconditions for at least about one day.

48. The pH stabilized anthocyanin extract composition of paragraph 44,wherein the stabilized anthocyanoside is stable at a pH of 7 for atleast about six hours.

49. The pH stabilized anthocyanin extract composition of paragraph 48,wherein the stabilized anthocyanoside maintained at a pH of 7 retains atleast 50% of the anthocyanin as a glycoside over about 4 hours.

50. A method to prepare a pH stabilized anthocyanin extract compositioncomprising the step of combining an anthocyanin extract and astabilizing compound having at least one —SH group, such that thecomposition is pH stable.

51. The method of paragraph 50, wherein the stabilizing compound isyeast extract, dihydrolipoic acid, derivatives of dihydrolipoic acid,cysteine, derivatives of cysteine, glutathione, derivatives ofglutathione, SH-proteinase, SH-metalloproteinase, peptides containingcysteine, peptides containing glutathioine, fermented oyster extract,thiolated chitosan, thiolated gelatin or mixtures thereof.

52. The method of either of paragraphs 50 or 51, wherein the anthocyaninextract is bilberry extract, blackcurrant extract, cranberry extract,black soybean extract, cowberry extract, blueberry extract or mixturesof two or more thereof.

53. The method of any of paragraphs 50 through 52, wherein the moleratio of stabilizing compound to anthocyanin extract is about 0.1 toabout 10.

54. The method of any of paragraph 50 through 53, wherein thecomposition is stabile toward degradation when exposed to an aqueousenvironment with a pH of between about 2 and about 12.

55. The method of any of paragraphs 50 through 54, wherein theanthocyanin extract comprises an anthocyanoside.

56. The method of paragraph 50, wherein the anthocyanin extract includesan anthocyanoside.

57. The method of paragraph 56, wherein the anthocyanoside is aglycoside of perlargonidin, peonidin, cyanidin, malvidin, petunidin,delphinidin or mixtures of two or more thereof.

58. The method of paragraph 50, wherein the stabilized anthocyaninextract is stabile at ambient conditions for at least about one day.

59. The method of paragraph 55, wherein the stabilized anthocyanoside isstable at a pH of 7 for at least about six hours.

60. The method of paragraph 59, wherein the stabilized anthocyanosidemaintained at a pH of 7 retains at least 50% of the anthocyanin as aglycoside over about four hours.

61. A method of providing a therapeutically beneficial amount of astabilized anthocyanin composition to a subject, comprising the step ofadministering to a subject a therapeutically beneficial amount of astabilized anthocyanin composition as described in any of paragraphs 1through 16 or paragraphs 39 through 49.

62. A method to treat arthrosclerosis comprising the step ofadministering a therapeutically effective amount of a stabilizedanthocyanin composition as described in any of paragraphs 1 through 16or paragraphs 39 through 49 to a subject.

63. A method to increase or maintain intracellular antioxidantconcentration of an anthocyanin, comprising the step of administering atherapeutically effective amount of a stabilized anthocyanin compositionas described in any of paragraphs 1 through 16 or paragraphs 39 through49.

64. A method to alleviate or reduce pain in a subject comprising thestep of administering a therapeutically effective amount of a stabilizedanthocyanin composition as described in any of paragraphs 1 through 16or paragraphs 39 through 49.

65. The stabilized anthocyanin extract of any of paragraphs 4, 12 or 42,wherein the mole ratio of stabilizing compound to anthocyanin extract isabout 0.5 to about 5.

66. The stabilized anthocyanin extract of any of paragraphs 4, 12 or 42,wherein the mole ratio of stabilizing compound to anthocyanin extract isabout 1 to about 1.

67. The stabilized anthocyanin extract of any of paragraphs 1 through 16or 39 through 49, wherein said stabilized anthocyanin extract is in theform of a pill, tablet, powder, granule, pellicle, ointment, cream,paste, solution, mixture, syrup, mucilage, emulsion, tincture, spirit,paint, drops or decoction.

68. The stabilized anthocyanin extract of any of paragraphs 1 through 16or 39 through 49, further comprising a physiologically acceptableadjuvant.

69. The stabilized anthocyanin extract of paragraph 68, wherein theadjuvant is a diluent, binder, disintegrating agent, lubricant, base,flavoring agent, sweetening agent, coloring agent, preservative,antioxidant, coating materials, film-forming materials, solvent,solubilizer, wetting agent, absorbent, filtering aid, emulsifying agent,surfactant, suspending agent, viscosity increasing agent, plasticizer,chelating agent, aerosol propellant, foaming agent, acidifying agent,alkalizing agent, buffering agent or mixtures thereof.

70. The method of any of paragraphs 20, 31 or 53, wherein the mole ratioof stabilizing compound to anthocyanin extract is about 0.5 to about 5.

71. The method of any of paragraphs 20, 31 or 53, wherein the mole ratioof stabilizing compound to anthocyanin extract is about 1 to about 1.

72. The stabilized anthocyanin extract of either of paragraphs 65 or 66,wherein the derivative of cysteine is N-acetylcysteine and theSH-proteinase is papain, bromelain, ficin, ehymopapain or mixturesthereof.

73. A method for stabilizing an anthocyanin-rich extract, comprising thestep:

contacting an anthocyanin-rich extract with at least one stabilizingcompound having at least one —SH group, wherein the stabilizing compoundcan be contacted with the anthocyanin at any time during the extractionprocess.

74. The method according to paragraph 73, wherein the compound having atleast one —SH group is yeast extract, dihydrolipoic acid, cysteine,glutathione or mixtures thereof.

75. The method of paragraph 73, wherein the mole ratio of thestabilizing compound having at least one —SH group to the anthocyanincompound is between about 0.1 and about 10.

76. The method of paragraph 75, wherein the mole ratio is between about0.5 and about 5.

77. The method of paragraph 75, wherein the mole ratio is about 1.

78. The method of any of paragraphs 73 through 77, wherein the compoundhaving at least one —SH group is added to the extraction solvent priorto contact with the anthocyanin-rich extract.

79. The method of any of paragraph 73 through 77, wherein the compoundhaving at least one —SH group is added to the extraction solvent afterthe anthocyanin-rich extract is combined with an extraction solvent.

80. A method to treat age-related macular degeneration (AMD), acataract, or retinopathy comprising the step of administering atherapeutically effective amount of a stabilized anthocyanin compositionas described in any of paragraphs 1 through 16 or paragraphs 39 through49 to a subject in need thereof.

81. A stabilized bilberry composition comprising bilberry extract andcysteine.

82. The stabilized bilberry composition of paragraph 81 wherein theratio of bilberry extract to cysteine is about 10 to about 1 on a weightbasis.

83. The stabilized bilberry composition of either of paragraphs 81 or82, wherein the composition is spray dried.

84. The stabilized bilberry composition of any of paragraphs 81 through83, wherein the stabilized composition retains at least about 80% of theoriginal anthocyanosides for a period of at least about 6 months asdetermined by HPLC analysis.

85. A method to increase the bioavailability of an anthocyanincomposition comprising the step of providing to a subject an anthocyanincomposition comprising an anthocyanin and a compound having at least one—SH group, wherein the amount of anthocyanin is increased in the subjectby a least twice the amount in comparison to a sample of an anthocyanincomposition devoid of a compound having at least one —SH group.

86. The method of paragraph 85, wherein the anthocyanin material is anextract.

87. The method of paragraph 86, wherein the extract is bilberry extract.

88. The method of paragraph 85, wherein the ratio of anthocyaninmaterial to the compound having at least one —SH group is between about10:0.1 and about 1:1 based on a weight basis.

89. The method of paragraph 87, wherein the ratio of anthocyanin in thebilberry extract to the compound having at least one —SH group isbetween about 10 to about 1.

90. The method of paragraph 85, wherein the plasma concentration of theanthocyanin in the presence of cysteine post four hours ofadministration is at least twice the amount of a plasma concentration ofanthocyanin absent the presence of the compound having at least one —SHgroup.

91. The method of any of paragraphs 85 through 90, wherein the compoundhaving at least one —SH group is yeast, dihydrolipoic acid, derivativesof dihydrolipoic acid, cysteine, derivatives of cysteine, glutathione,derivatives of glutathione, SH-proteinase, SH-metalloproteinase,peptides containing cysteine, peptides containing glutathione, fermentedoyster extract, thiolated chitosan, thiolated gelatin or mixturesthereof.

The following examples are not to be meant as limiting but are presentedto provide additional information and support for the invention.

The present exemplary section relates to bilberry extract and blackcurrant extract, as shown in the table below:

Producer Lot. No. Bilberry extract Omya-Peralta GmbH BB0823-1 Blackcurrant extract Omya-Peralta GmbH BC6006

EXAMPLE 1

Sample a) 60 mg bilberry extract [37% anthocyanosides] (0.048 mmolanthocyanosides expressed as cyanidin-3-O-glucoside) were added to a 100ml flask and filled to volume with sodium phosphate buffer (5% [w/w],pH=7.0), and stirred until dissolution was complete. A 1 ml sample wastaken immediately and acidified with formic acid to pH=1.0 and analyzedby HPLC for the content of anthocyanosides (Fresh Sample). The remainingsolution was kept for 4 hours at 37° C. (water bath) with stirring.Thereafter another sample (Blank sample), representing unprotecteddegradation, was taken and acidified.

Sample b) 20 mg reduced L-glutathione (0.065 mmol) were added to a 100ml flask with 60 ml sodium phosphate buffer (5% [w/w], pH=7.0) andstirred until dissolution was complete. 60 mg bilberry extract [37%anthocyanosides] (0.048 mmol expressed as cyanidin-3-O-glucoside) werethen added and the flask was filled to volume with sodium phosphatebuffer (5% [w/w], pH=7.0) and kept for 4 hours at 37° C. (water bath)with stirring.

Sample c) 30 mg dihydrolipoic acid (0.144 mmol) were added to a 100 mlflask with 60 ml sodium phosphate buffer (5% [w/w], pH=7.0) and stirreduntil dissolution was complete. 60 mg bilberry extract [37%anthocyanosides] (0.048 mmol expressed as cyanidin-3-O-glucoside) werethen added and the flask was filled to volume with sodium phosphatebuffer (5% [w/w], pH=7.0) and kept for 4 hours at 37° C. (water bath)with stirring.

After 4 hours, samples were taken and acidified immediately with formicacid to pH=1.0 and analyzed by HPLC for the content of anthocyanosides.Degradation is expressed as decrease in peak area of individual peaks(calculated as % left after 4 hours).

From the table below it was concluded that anthocyanosides aresubstantially more stable at pH=7.0 at 37° C. in presence of either DHLAor GSH when compared to blank (unprotected) samples. After 4 hours, theblank sample showed that about 25% of residual anthocyanosides wereobserved whereas the protected samples yielded more than 60% residualanthocyanosides. The protective efficacy of 30 mg DHLA is comparable to20 mg GSH. The protective efficacy seems to be related to the basicanthocyanin-skeleton exemplified by delphinidin glycosides which areless well protected when compared to cyanidin glycosides. For eachindividual anthocyanoside tested, a protective effect, e.g. moreresidual anthocyanosides when compared to blank samples, was observed.

TABLE Peak Area of anthocyanosides after incubation for 4 hours at 37°C. Blank (4 hours) DHLA (4 hours) GSH (4 hours) Peak ID. Fresh (0 hours)Area % left Area % left Area % left Dp-3O-Gal 654.4 50.9 7.8 395.8 60.5331.3 50.6 Dp-3O-Glc 726.6 46.3 6.4 414.8 57.1 350.8 48.3 Cn-3O-Gal465.8 191.1 41.0 300.3 64.5 360.2 77.3 Dp-3O-Ara 564.9 39.1 6.9 364.464.5 283.6 50.2 Cn-3O-Glc 987.9 410.3 41.5 671.1 67.9 797.5 80.7Pt-3O-Gal 216.4 40.7 18.8 132.8 61.4 150.0 69.3 Cn-3O-Ara 369.6 142.338.5 258.4 70.2 293.7 79.5 Pt-3O-Glc 473.4 78.8 16.6 271.9 57.4 315.666.7 Pe-3O-Gal 47.6 21.0 44.1 32.7 68.7 36.3 76.3 Pt-3O-Ara 140.6 22.816.2 93.7 66.6 97.6 69.4 Pe-3O-Glc 223.6 94.8 42.4 139.2 62.3 172.0 76.9Mv-3O-Gal 179.1 67.3 37.6 112.5 62.8 133.2 74.4 Pe-3O-Ara 23.5 9.2 39.121.4 91.1 18.4 78.3 Mv-3O-Glc 455.9 164.7 36.1 287.4 63.0 336.2 73.7Mv-3O-Ara 108.3 36.8 34.0 75.2 69.4 81.2 75.0 Total 5637.6 1416.1 25.13572.6 63.4 3757.6 66.7 Dp . . . Delphinidin, Cn . . . Cyanidin, Pt . .. Petunidin, Pe . . . Peonidin, Mv . . . Malvidin, Gal . . . Glactoside,Glc . . . Glucoside, Ara . . . Arabinoside

Samples were prepared as outlined below in HPLC Analytical Method. Teststability samples were immediately acidified (to block degradation) andinjected undiluted following the detailed description provided below.

EXAMPLE 2

Sample a) 60 mg bilberry extract [37% anthocyanosides] (0.048 mmolexpressed as cyanidin-3-O-glucoside) were added to a 100 ml, filled tovolume with sodium phosphate buffer (5% [w/w], pH=7.0) and stirred untildissolution was complete. A 1 ml sample was immediately taken andacidified with formic acid to pH=1.0 and analyzed by HPLC for thecontent of anthocyanosides. The remaining solution was kept at 37° C.(water bath) with stirring and additional 1 ml samples (Blank Samples),representing unprotected degradation, were sampled every 15 minutes. Thesamples were analyzed for selected anthocyanosides by HPLC.

Sample b) 20 mg reduced L-glutathione (0.065 mmol) were added to a 100ml flask with 60 ml sodium phosphate buffer (5% [w/w], pH=7.0) andstirred until dissolution was complete. 60 mg bilberry extract [37%anthocyanosides] (0.048 mmol expressed as cyanidin-3-O-glucoside) wereadded and the flask was filled to volume with sodium phosphate buffer(5% [w/w], pH=7.0) and kept at 37° C. (water bath) with stirring. Every15 minutes, 1 ml samples were taken and analyzed for selectedanthocyanosides by HPLC.

From the tables below it was concluded that anthocyanosides aresubstantially more stable at pH=7.0 and 37° C. in presence of GSH whencompared to blank samples. The protective activity started immediatelyafter dissolution and lasts for at least 4 hours as indicated by thecomparative decay of the selected anthocyanosides over time. Again, theprotective effect is believed to be related to the anthocyanin-skeletonexemplified by 65% residual delphinidin-3-O-Galactose, 67% residualpetunidin-3-O-Galactose and 77% residual cyanidin-3-O-galactose afterincubation for 4 h 30 min. For each individual anthocyanosides tested aprotective effect, e.g. more residual anthocyanosides when compared toblank samples, was observed.

TABLE Peak Area of anthocyanosides after incubation at 37° C. (BlankSample) Incubation Time Dp-3O-Gal Cn-3O-Gal Pt-3O-Gal (hours) (PeakArea) (Peak Area) (Peak Area) 0:00 699.51 498.85 237.44 0:15 547.09390.37 179.60 0:30 603.54 433.94 203.39 0:45 451.09 347.29 152.46 1:00467.23 380.25 161.49 1:15 354.36 305.40 124.16 1:30 — — — 1:45 401.11353.94 144.59 2:00 356.25 337.90 132.38 2:15 320.33 320.70 121.96 2:30262.70 271.19 96.36 2:45 264.07 274.22 99.38 3:00 288.65 324.58 127.473:15 289.95 324.25 123.02 3:30 237.41 281.19 98.72 3:45 — — — 4:00243.90 315.54 107.51 4:15 226.99 300.50 102.44 4:30 135.48 205.36 58.78— . . . No sample available

TABLE Peak Area of anthocyanosides after incubation at 37° C. (GSHProtected Sample) Incubation Time Dp-3O-Gal Cn-3O-Gal Pt-3O-Gal (hours)(Peak Area) (Peak Area) (Peak Area) 0:00 731.12 508.09 248.16 0:15739.50 504.88 245.04 0:30 674.83 470.61 222.35 0:45 598.57 425.89 195.631:00 652.53 462.71 213.84 1:15 648.70 452.17 210.34 1:30 618.45 431.86202.34 1:45 609.47 442.86 194.88 2:00 583.66 436.19 193.23 2:15 592.13442.30 199.06 2:30 528.71 390.23 169.36 2:45 560.24 422.32 183.97 3:00562.10 433.21 189.41 3:15 562.39 425.54 185.92 3:30 553.82 422.54 181.533:45 495.67 397.72 173.03 4:00 563.95 446.42 193.79 4:15 540.10 419.80179.46 4:30 531.62 420.37 181.46 4:45 478.52 393.21 166.84

TABLE %-residual anthocyanosides after incubation at 37° C. for 4.30hours Dp-3O-Gal Cn-3-O-Gal Pt-3-O-Gal (% left) (% left) (% left) Blank19% 41% 25% GSH-protected 65% 77% 67%Samples were prepared as outlined below in HPLC Analytical Method. Teststability samples were immediately acidified (to block degradation) andinjected undiluted following the detailed description provided below.

EXAMPLE 3

Sample a) 60 mg black currant extract [35% anthocyanosides] (0.037 mmolanthocyanosides expressed as cyanidin-3-O-rutinoside) were added to a100 ml flask and filled to volume with sodium phosphate buffer (5%[w/w], pH=7.0) and stirred until dissolution was complete. A 1 ml samplewas taken immediately, acidified with formic acid to pH=1.0 and analyzedby HPLC for the content of anthocyanosides (Fresh Sample). The remainingsolution was kept for 4 hours at 37° C. (water bath) with stirring.Thereafter samples (Blank sample), representing unprotected degradation,were taken and acidified.

Sample b) 20 mg reduced L-glutathione (0.065 mmol) were added to a 100ml flask with 60 ml sodium phosphate buffer (5% [w/w], pH=7.0) andstirred until dissolution was complete. 60 mg black currant extract [35%anthocyanosides] (0.037 mmol anthocyanosides expressed ascyanidin-3-O-rutinoside) were then added and the flask was filled tovolume with sodium phosphate buffer (5% [w/w], pH=7.0) and kept for 4hours at 37° C. (water bath) with stirring.

Sample c) 20 mg dihydrolipoic acid (0.096 mmol) were added to a 100 mlflask with 60 ml sodium phosphate buffer (5% [w/w], pH=7.0) and stirreduntil dissolution was completed. 60 mg black currant extract [35%anthocyanosides] (0.037 mmol anthocyanosides expressed ascyanidin-3-O-rutinoside) were added to the flask, filled to volume withsodium phosphate buffer (5% [w/w], pH=7.0) and kept for 4 hours at 37°C. (water bath) with stirring.

Sample d) 20 mg L-cysteine acid (0.165 mmol) were added to a 100 mlflask with 60 ml sodium phosphate buffer (5% [w/w], pH=7.0) and stirreduntil dissolution was complete. 60 mg black currant extract [35%anthocyanosides] (0.037 mmol anthocyanosides expressed ascyanidin-3-O-rutinoside) were added to the flask, filled to volume withsodium phosphate buffer (5% [w/w], pH=7.0) and kept for 4 hours at 37°C. (water bath) with stirring.

After 4 hours samples were taken, acidified immediately with formic acidto pH=1.0 and analyzed by HPLC for the content of anthocyanosides.Degradation is expressed as decrease in peak area of individual peaks(calculated as % left after 4 hours).

From the table below it was determined that anthocyanosides aresubstantially more stable at pH=7.0 and 37° C. in presence of DHLA, GSHor L-cysteine when compared to blank samples. After 4 hours, in theblank sample between 9.5-33.4% of residual anthocyanosides were observedwhereas the GSH protected samples yielded 50.4-65.0% residualanthocyanosides. Comparative figures for DHLA were 36.7-38.9%. For eachindividual anthocyanosides tested a protective effect, e.g. moreresidual anthocyanosides when compared to blank samples, was observed.

TABLE Peak Area of anthocyanosides after incubation for 4 hours at 37°C. Fresh GSH 20 mg L-cysteine solution Blank DHLA 20 mg % 20 mg Peak IDArea Area % left Area % left Area left Area % left Dp-3-O-Glc 1481 140.89.5 770.7 52.0 745.6 50.4 543.0 36.7 Dp-3-O-Rut 5207 626.2 12.0 269251.7 2764 53.1 1975 38.0 Cn-3-O-Glc 491.1 154.0 31.4 254.1 51.7 315.764.3 186.8 38.0 Cn-3-O-Rut 3539 1183 33.4 1809 51.1 2299 65.0 1377 38.9Dp . . . Delphinidin, Cn . . . Cyanidin, Glc . . . Glucoside, Rut . . .Rutinoside

Samples were prepared as outlined below in HPLC Analytical Method. Teststability samples were immediately acidified (to block degradation) andinjected undiluted following the detailed description provided below.

EXAMPLE 4

Sample a) 60 mg black currant extract [35% anthocyanosides] (0.037 mmolanthocyanosides expressed as cyanidin-3-O-rutinoside) were added to a100 ml flask, filled to volume with sodium phosphate buffer (5% [w/w],pH=7.0) and stirred until dissolution was complete. The solution waskept for 4 hours at 37° C. (water bath) with stirring. Thereafter asample (Blank sample), representing unprotected degradation, was takenand acidified.

Samples b-e) 5, 10, 20 or 60 mg dihydrolipoic acid (0.024-0.288 mmol)were added to 100 ml flasks with 60 ml sodium phosphate buffer (5%[w/w], pH=7.0) and stirred until dissolution was complete. To each flaskwas added 60 mg black currant extract [35% anthocyanosides] (0.037 mmolanthocyanosides expressed as cyanidin-3-O-rutinoside). The flasks werefilled to volume with sodium phosphate buffer (5% [w/w], pH=7.0) andkept for 4 hours at 37° C. (water bath) with stirring.

Samples f-i) 5, 10, 20 or 60 mg GSH (0.016-0.192 mmol) were added to 100ml flasks with 60 ml sodium phosphate buffer (5% [w/w], pH=7.0) andstirred until dissolution was complete. To each flask was added 60 mgblack currant extract [35% anthocyanosides] (0.037 mmol anthocyanosidesexpressed as cyanidin-3-O-rutinoside). The flasks were filled to volumewith sodium phosphate buffer (5% [w/w], pH=7.0) and kept for 4 hours at37° C. (water bath) with stirring.

Samples j-m) 5, 10, 20 or 60 mg L-cysteine (0.041-0.492 mmol) were addedto 100 ml flasks with 60 ml sodium phosphate buffer (5% [w/w], pH=7.0)and stirred until dissolution was complete. To each flask was added 60mg black currant extract [35% anthocyanosides] (0.037 mmolanthocyanosides expressed as cyanidin-3-O-rutinoside). The flasks werefilled to volume with sodium phosphate buffer (5% [w/w], pH=7.0) andkept for 4 hours at 37° C. (water bath) with stirring.

After 4 hours, samples were taken, acidified immediately with formicacid to pH=1.0 and analyzed by HPLC for the content of anthocyanosides.Degradation is expressed as a decrease in the sum peak area for 4 leadanthocyanosides (Dp-3-O-Glc, Dp-3-O-Rut, Cn-3-O-Glc, Cn-3-O-Rut),calculated as % left after 4 hours.

From the tables below it was determined that anthocyanosides weresubstantially protected by DHLA, GSH or L-cysteine in a dose-dependentmanner.

TABLE Peak Area of anthocyanosides after incubation for 4 hours at 37°C. DHLA added Sum Peak Area Sum Peak Area (mg) Blank Sample ProtectedSample 5 2103 3503 10 2103 3615 20 2103 3677 60 2103 3973

TABLE Peak Area of anthocyanosides after incubation for 4 hours at 37°C. GSH added Sum Peak Area Sum Peak Area (mg) Blank Sample ProtectedSample 5 2103 2939 10 2103 3314 20 2103 4036 60 2103 4204

TABLE Peak Area of anthocyanosides after incubation for 4 hours at 37°C. L-cysteine Sum Peak Area Sum Peak Area added (mg) Blank SampleProtected Sample 5 2103 3119 10 2103 3625 20 2103 4190 60 2103 4202

Samples were prepared as outlined below in HPLC Analytical Method. Teststability samples were immediately acidified (to block degradation) andinjected undiluted following the detailed description provided below.

EXAMPLE 5

Sample a) 60 mg black currant extract [35% anthocyanosides] (0.037 mmolanthocyanosides expressed as cyanidin-3-O-rutinoside) were added to a100 ml flask, filled to volume with sodium phosphate buffer (5% [w/w],pH=7.0) and stirred until dissolution was complete. A 1 ml sample wastaken immediately, acidified with formic acid to pH=1.0 and analyzed byHPLC for the content of anthocyanosides. The remaining solution was keptat 37° C. (water bath) with stirring and 1 ml samples (Blank Samples),representing unprotected degradation, were taken every 15 minutes. Thesamples were analyzed for selected anthocyanosides by HPLC.

Sample b) 20 mg reduced L-glutathione (0.065 mmol) were added to a 100ml flask with 60 ml sodium phosphate buffer (5% [w/w], pH=7.0) andstirred until dissolution was complete. 60 mg black currant extract [35%anthocyanosides] (0.037 mmol anthocyanosides expressed ascyanidin-3-O-rutinoside) were added, the flask was filled to volume withsodium phosphate buffer (5% [w/w], pH=7.0) and kept for at 37° C. (waterbath) with stirring. Every 15 minutes 1 ml samples were taken andanalyzed for selected anthocyanosides by HPLC.

From the tables below it could was determined that anthocyanosides aresubstantially more stable at pH=7.0 and 37° C. in presence of GSH whencompared to blank samples. The protective activity starts immediatelyafter dissolution and lasts for at least 4 hours as indicated by thecomparative decay of the anthocyanosides over time. The protectionefficacy may be dependent on the anthocyanin-skeleton, exemplified bythe improved protection of cyanidin-glycosides when compared todelphinidin-glycosides. However, when comparing the blank to theprotected sample, delphinidin-glycosides were substantially betterprotected than cyaniding-glycosides.

For each individual anthocyanosides tested a protective effect, e.g.more residual anthocyanosides when compared to blank samples, wasobserved.

TABLE Peak Area of anthocyanosides after incubation at 37° C. (Blanksample) Incubation Time Dp-3O-Glc Dp-3-O-Rut Cn-3-O-Glc Cn-3-O-Rut(hours) (Peak Area) (Peak Area) (Peak Area) (Peak Area) 0:00 1480.75207.5 491.1 3539.1 0:15 969.6 3604.2 368.9 2707.4 0:30 907.9 3366.5361.9 2620.7 0:45 745.2 2787.9 321.0 2342.2 1:00 702.1 2660.6 312.02278.1 1:30 605.4 2299.5 293.4 2130.7 1:45 599.9 2291.6 283.8 2104.92:00 465.1 1825.7 256.7 1894.9 2:15 449.8 1747.2 250.3 1851.7 2:30 394.01552.5 243.9 1790.9 2:45 362.7 1442.9 226.8 1694.9 3:00 304.8 1235.4213.7 1573.5 3:15 285.1 1166.0 204.9 1531.6 3:30 208.8 907.2 180.11370.4 3:45 214.5 891.1 181.6 1361.3 4:00 140.8 626.2 154.0 1182.9

TABLE Peak Area of anthocyanosides after incubation at 37° C. (GSHprotected sample) Incubation Dp-3O-Glc Dp-3-O-Rut Cn-3-O-Glc Cn-3-O-RutTime (hours) (Peak Area) (Peak Area) (Peak Area) (Peak Area) 0:00 1464.05201.3 480.6 3473.6 0:15 1280.4 4584.2 426.1 3108.1 0:45 1209.3 4275.7418.6 2964.3 1:00 1219.0 4273.1 423.8 3015.3 1:15 1178.1 4168.7 415.32958.5 1:45 1051.8 3749.9 376.4 2688.8 2:00 1077.0 3813.3 387.6 2777.82:15 981.9 3544.9 356.5 2579.6 2:45 876.8 3199.5 337.5 2447.1 3:00 917.23327.2 349.5 2534.7 3:15 812.5 2993.9 325.6 2366.8 3:45 742.7 2761.0312.6 2274.2 4:00 745.6 2763.6 315.7 2298.9 4:15 682.0 2579.8 300.32199.7

TABLE %-residual anthocyanosides after incubation at 37° C. for 4 hoursDp-3O-Glc Dp-3-O-Rut Cn-3-O-Glc Cn-3-O-Rut (% left) (% left) (% left) (%left) Blank 10% 12% 31% 33% GSH-protected 51% 53% 66% 66%

Samples were prepared as outlined below in HPLC Analytical Method. Teststability samples were immediately acidified (to block degradation) andinjected undiluted following the detailed description provided below.

EXAMPLE 6

Sample a) 5 mg delphinine chloride (0.015 mmol) was transferred into a100 ml flask, dissolved in 1 ml methanol and filled to volume withsodium phosphate buffer (5% [w/w], pH=7.0). The sample was kept withstirring at 37° C. Samples were taken immediately after dissolution,after 15 minutes, 1, 2, and 3 hours of incubation. The samples wereacidified with formic acid to pH=1.0 and analyzed by HPLC.

Sample b) 5 mg malvidin chloride (0.014 mmol) was transferred into a 100ml flask, dissolved in 1 ml methanol and filled to volume with sodiumphosphate buffer (5% [w/w], pH=7.0). The sample was kept with stirringat 37° C. Samples were taken immediately after dissolution and at 1, 2,and 3 hours of incubation. The samples were acidified with formic acidto pH=1.0 and analyzed by HPLC.

Sample c) 5 mg delphinidine chloride (0.015 mmol) and 10 mg GSH (0.032mmol) were transferred into a 100 ml flask, dissolved in 1 ml methanoland filled to volume with sodium phosphate buffer (5% [w/w], pH=7.0).The sample was kept with stirring at 37° C. Samples were taken at 15minutes after dissolution and after 1, 2 and 3 hours of incubation. Thesamples were acidified with formic acid to pH=1.0 and analyzed by HPLC.

Sample d) 5 mg malvidin chloride (0.014 mmol) and 10 mg GSH (0.032 mmol)were transferred into a 100 ml flask, dissolved in 1 ml methanol andfilled to volume with sodium phosphate buffer (5% [w/w], pH=7.0). Thesample was kept with stirring at 37° C. Samples were taken 15 minutesafter dissolution and after 1, 2 and 3 hours of incubation. The sampleswere acidified with formic acid to pH=1.0 and analyzed by HPLC.

From the table below it was determined that GSH did not protect well thefree anthocyanin skeleton. The molar ratio between GSH and delphinidinor malvidin is higher than 2:1 indicating that the lack of activity isnot related to a low concentration of GSH. The decay of the freeanthocyanin is almost unaffected by the presence of GSH which is incontrast to the observations using anthocyanin-glycosides bearing thesame skeleton.

TABLE Peak Area of delphinidin and malvidin after incubation at 37° C.Time Blank GSH 10 mg (hours) Delphinidin Malvidin Delphinidin Malvidin 048.6 1228 NA NA 0.25 BLD NA BLD NA 1 BLD 661.6 BLD 530.1 2 BLD 175.3 BLD263.5 3 BLD 56.4 BLD 5.6 BLD . . . Below Limit of Detection, NA . . .not applicable

Samples were prepared as outlined below in HPLC Analytical Method. Teststability samples were immediately acidified (to block degradation) andinjected undiluted following the detailed description provided below.

EXAMPLE 7

Sample a) 60 mg bilberry extract [37% anthocyanosides] (0.048 mmolanthocyanosides expressed as cyanidin-3-O-glucoside) was added to a 100ml flask, filled to volume with sodium phosphate buffer (5% [w/w], pHadjusted to the value given below) and stirred until dissolution wascomplete. The solution was kept for 4 hours at 37° C. (water bath) withstirring. Thereafter a sample (Blank sample), representing unprotecteddegradation, was taken and acidified to pH=1.0 with formic acid.

Sample b) 20 mg reduced L-glutathione (0.065 mmol) was added to a 100 mlflask with 60 ml sodium phosphate buffer (5% [w/w], pH adjusted to thevalue given below) and stirred until dissolution was complete. 60 mgbilberry extract [37% anthocyanosides] (0.048 mmol expressed ascyanidin-3-O-glucoside) was added to the flask, filled to volume withsodium phosphate buffer (5% [w/w], pH=7.0) and kept for 4 hours at 37°C. (water bath) with stirring.

After 4 hours samples were taken and acidified immediately with formicacid to pH=1.0 and analyzed by HPLC for the content of anthocyanosides.Degradation is expressed as decrease in the sum peak area for 4anthocyanosides, calculated as % left after 4 hours.

From the table below it was determined that GSH protects anthocyanosidesespecially in the pH-range where anthocyanosides are susceptible todegradation (pH>5). Not surprisingly, at lower pH-values whereanthocyanosides are stable by itself the protective effect isdiminished.

TABLE %-residual anthocyanosides after incubation at 37° C. for 4 hours% residual anthocyanosides pH value Blank Sample 20 mg GSH 3.0 87.3 97.05.0 81.9 86.5 7.0 25.1 66.7 9.0 8.8 40.5 10.0 4.0 18.8 11.0 BLD 3.1

Samples were prepared as outlined below in HPLC Analytical Method. Teststability samples were immediately acidified (to block degradation) andinjected undiluted following the detailed description provided below.

The protection effect is a parameter that is calculated by dividing theresidual ratio of the protected sample by the residual ratio of a blanksample (with bilberry extract at selected pH values without reducedglutathione). The difference thus demonstrates the effect of reducedL-glutathione (GSH) of the stability of anthocyanins of bilberry extractover a pH range of between about 3 and about 11.

It was noted that when the pH value increased, the degradation of theextract increased. In all pH values tested, the L-glutathione (GSH)protected the anthocyanins. Additionally, the Protection Effectincreased with pH values. In alkaline environments, the GSH had agreater stabilizing effect as seen in the table above and in FIGS. 19and 20.

EXAMPLE 8

Sample a) 60 mg GSH (0.192 mmol) were placed into a 100 ml flask,dissolved and filled up to volume with sodium phosphate buffer (5%[w/w], pH=7.0). The sample was kept with stirring at 37° C. Samples weretaken immediately after dissolution and after 1, 2, 3 and 4 hours ofincubation. The samples were analyzed by HPLC for GSH and GSSG(representing the oxidized form of GSH).

Sample b) 60 mg reduced L-glutathione (0.192 mmol) were added to a 100ml flask with 60 ml sodium phosphate buffer (5% [w/w], pH=7.0) andstirred until dissolution was complete. 60 mg bilberry extract [37%anthocyanosides] (0.048 mmol expressed as cyanidin-3-O-glucoside) wereadded, the flask was filled to volume with sodium phosphate buffer (5%[w/w], pH=7.0) and kept at 37° C. (water bath) with stirring. Sampleswere taken immediately after dissolution and after 1, 2, 3 and 4 hoursof incubation. The samples were analyzed by HPLC for GSH and GSSG(representing the oxidized form of GSH).

From the tables below it was determined that GSH was oxidized to GSSG inaqueous buffer solution (pH=7.0) at 37° C. Addition of bilberry extractaccelerated the oxidation of GSH to GSSG by a factor about 2 to about 3suggesting that bilberry extract serves as partner for a redox reaction.Based on this result it was evident that the couple GSH/GSSG has aconsiderably low redox potential yielding oxidized GSSG and reducedanthocyanosides.

TABLE Peak Area of GSH and GSSG and peak area ratio after incubation inbuffer pH = 7.0 at 37° C. GSH Sample Incubation Peak Area Peak Area Time(hours) GSH Ratio % GSSG Ratio % 0 4760 92.3 394.9 7.7 1 4744 92.0 511.59.9 2 4220 81.9 763.2 14.8 3 4053 78.6 1051 20.4 4 3777 73.3 1209 23.5

TABLE Peak Area of GSH and GSSG and peak area ratio after incubation inbuffer pH = 7.0 at 37° C. in presence of bilberry extract GSH + bilberrySample Incubation Peak Area Peak Area Time (hours) GSH Ratio % GSSGRatio % 0 4760 92.3 394.9 7.7 1 4071 79.0 1260 24.4 2 3378 65.5 172233.4 3 3045 59.1 2033 39.4 4 2461 47.7 2447 47.5

Samples were prepared as outlined below in HPLC Analytical Method. Teststability samples were immediately acidified (to block degradation) andinjected undiluted following the detailed description provided below.

Stability of Black Currant Extract

120 mg black currant extract/100 ml solution were incubated with 30 mgglutathione (GSH)/100 ml in buffered solution (pH=7.0) as the incubationmedium used for CaCo-2 absorption tests.

These investigations provide information on the stability/degradation ofselected black currant lead-anthocyanosides in analytical solutions andin the incubation medium used for the CaCo-2 test in presence ofglutathione.

Delphindin-3-O-glucoside, (Dp-Glc), Delphinidin-3-O-rutinoside (Dp-Rut)and Cyanidin-3-O-rutinoside (Cn-Rut) were chosen for analysis.

These investigations showed that both delphinidin-glycosidesinvestigated were more susceptible to degradation thancyanidin-rutinoside. Dp-glc was more susceptible than Dp-rut under thegiven conditions. All anthocyanosides investigated were slightly morestable in incubation medium resembling the ileal fluid than the bufferedsolution (See FIGS. 13 and 14). Most likely, the increase in stabilityis caused by the presence of putatively stabilizing ingredients in thecomplex medium (eventually re-cycling glutathione).

CaCo-2 cells were incubated with 120 mg black currant extract and 30 mgglutathione/100 ml medium at 37° C. for up to 2 hours. At 30, 60 and 120minutes, 3 wells were processed for analysis by collection of theincubation medium and extraction of the anthocyanosides absorbed intothe cells.

As seen in FIG. 15, all 3 anthocyanosides investigated were absorbedinto CaCo-2 cells. The highest absorption was observed for Cn-Rut after60 minutes of incubation. At this time point the recovery ofanthocyanosides in CaCo-2 cells amounts to 2.5% of the concentrationdetermined in the supernatant incubation medium. Most interestingly, the% uptake resembles the stability of the anthocyanosides in bufferedmedium and incubation medium (without cells). It was further observedthat for all anthocyanosides the maximum absorption is seen after 60minutes of incubation.

60 mg bilberry extract ±30 mg glutathione/100 ml cell-free incubationmedia were held at 37° C. for 1 hour. Samples were analyzed prior orafter the incubation period for 15 anthocyanosides present in bilberryextract.

This investigation provided information on the basic stability of theanthocyanosides in incubation medium (pH=7.0) at the conditions appliedduring CaCo-2 testing. Secondly, the effect of glutathione on thestability is elucidated.

As seen in FIG. 16, all anthocyanosides were stabilized by glutathione.The most pronounced stabilization was observed for Dp-glycosides. Thisis of importance because Dp-glycosides are the most susceptible bilberryglucosides in terms of degradation at pH=7.0. From this analyticalinvestigation it was determined that glutathione protectsanthocyanosides from degradation at pH=7.0. Moreover it is noted thatglutathione protects all anthocyanidin-structures present in bilberry.

A detailed analysis of the protection reveals that a tendency betweenprotective potency and chemical structure of anthocyanidin exists. Dp,the most susceptible structure, is stabilized to the greatest extentwhereas the protective effect for the most stable structure (Cn) iscomparably low. In summary all anthocyanosides studied degraded lessthan 25% at pH=7.0 during 1 hour at 37° C. in presence of glutathione.

CaCo-2 cells were incubated with 60 mg bilberry extract with/without 30mg glutathione/100 m medium at 37° C. for 1 hour. Thereafter 3 wellswere processed for analysis by collection of the incubation medium andextraction of the anthocyanosides absorbed into the cells.

As seen in FIG. 17, all bilberry anthocyanosides studied were betterabsorbed in presence of glutathione. This points to a correlation of anincreased absorption with the stabilization of anthocyanosides withglutathione. The most likely explanation is that the degree ofabsorption of anthocyanosides is dependent on the concentration ofintact anthocyanosides in the incubation media. In other words, theabsorption of anthocyanosides may be dependent on a concentrationgradient between the outer and the inside of the cells (the higherconcentration outside, the higher the absorption). As glutathione wasshown to increase the stability of anthocyanosides outside the cells,absorption into the cells follows the theory proposed.

EXAMPLE 9

Sample a) 60 mg bilberry extract [37% anthocyanosides] (0.048 mmolanthocyanosides expressed as cyanidin-3-O-glucoside) were added to a 100ml flask with 60 ml sodium phosphate buffer (5% [w/w], pH=7.0), andstirred until dissolution was complete. A 1 ml sample was takenimmediately and acidified with formic acid to pH=1.0 and analyzed byHPLC for the content of anthocyanosides (Fresh Sample). The remainingsolution was kept for 4 hours at 37° C. (water bath) with stirring.Thereafter another sample (Blank sample), representing unprotecteddegradation, was taken and acidified.

Sample b) 20 mg L-cysteine (0.065 mmol) were added to a 100 ml flaskwith 60 ml sodium phosphate buffer (5% [w/w], pH=7.0) and stirred untildissolution was complete. 60 mg bilberry extract [37% anthocyanosides](0.048 mmol expressed as cyanidin-3-O-glucoside) were then added to theflask and stir to fully dissolve, then kept for 4 hours at 37° C. (waterbath) with stirring.

After 4 hours, samples were taken and acidified immediately with formicacid to pH=1.0 and analyzed by HPLC for the content of anthocyanosides.Degradation is expressed as decrease in peak area of individual peaks(calculated as % left after 4 hours).

From the table below it was concluded that anthocyanosides aresubstantially more stable at pH=7.0 at 37° C. in presence of L-cysteinewhen compared to blank (unprotected) samples. After 4 hours, the blanksample showed that about 25% of residual anthocyanosides were observedwhereas the protected samples yielded more than 65% residualanthocyanosides.

TABLE Peak Area of anthocyanosides after incubation for 4 hours at 37°C. Fresh solution Blank L-cysteine 20 mg Peak ID Area Area % left Area %left Dp-3-O-Gal 654.4 50.9 7.8 331.3 50.6 Dp-3-O-Glc 726.6 46.3 6.4350.8 48.3 Cn-3-O-Gal 465.8 191.1 41.0 360.2 77.3 Cn-3-O-Glc 987.9 410.341.5 797.5 80.7 Dp . . . Delphinidin, Cn . . . Cyanidin, Glc . . .Glucoside, Gal . . . Galactoside

Samples were prepared as outlined above in the HPLC Analytical Method.Test stability samples were immediately acidified (to block degradation)and injected undiluted following the detailed description providedbelow.

EXAMPLE 10

Sample a) 60 mg bilberry extract [37% anthocyanosides] (0.048 mmolanthocyanosides expressed as cyanidin-3-O-glucoside) were added to a 100ml flask and filled with 60 ml sodium phosphate buffer (5% [w/w],pH=7.0), and stirred until dissolution was complete. A 1 ml sample wastaken immediately and acidified with formic acid to pH=1.0 and analyzedby HPLC for the content of anthocyanosides (Fresh Sample). The remainingsolution was kept for 4 hours at 37° C. (water bath) with stirring.Thereafter another sample (Blank sample), representing unprotecteddegradation, was taken and acidified.

Samples b-e) 5, 10, 20 or 60 mg L-cysteine (0.041-0.492 mmol) were addedto 100 ml flasks with 60 ml sodium phosphate buffer (5% [w/w], pH=7.0)and stirred until dissolution was complete. To each flask was 60 mgbilberry extract [37% anthocyanosides] (0.048 mmol anthocyanosidesexpressed as cyanidin-3-O-glucoside). Then stir to dissolve the bilberryextract and kept for 4 hours at 37° C. (water bath) with stirring.

After 4 hours, samples were taken, acidified immediately with formicacid to pH=1.0 and analyzed by HPLC for the content of anthocyanosides.Degradation is expressed as a decrease in the sum peak area for allanthocyanosides, calculated as % left after 4 hours.

From the tables below it was determined that anthocyanosides weresubstantially protected by L-cysteine in a dose-dependent manner.

TABLE Peak Area of anthocyanosides after incubation for 4 hours at 37°C. L-cysteine Anthocyanoside left % Anthocyanoside left % added (mg)Blank Sample Protected Sample 5 25.2 51.8 10 25.2 60.2 20 25.2 68.7 6025.2 69.1

EXAMPLE 11

Materials

Bilberry capsules, provided by Omnica GmbH, sample No.: 20070601 and20070602.

Sample No. 20070601 only contained bilberry extract, as blank sample

Sample No. 20070602 contained bilberry extract and 10% cysteine

5% Sodium phosphate buffer

Analysis Method

Agilent 1100 HPLC, UV-VIS detector at 530 nm

Experiment

To a 100 ml flask was added 60.0 g 5% Sodium phosphate buffer (pH=7.0)and 60.0 mg powder of bilberry extract from a capsule. The solution wassonicated until all the solid were fully dissolved. A sample was takenimmediately and then the flask was placed into a 37° C. water bath andmaintained for 4 hours to determine the residual ratio of anthocyanins.

Result and Conclusion

HPLC Data

The HPLC areas were used as a measure of the content of the compounds.

The content did not include un-substituted anthocyanidin base

Comparison of the two capsules (20070601 and 20070602)

The following tables and FIGS. 21 through 26 provide evidence that theuse of cysteine helps to stabilize bilberry extract.

Fresh Content Final Content Batch No. Time zero 4 hours Residual Ratio %20070601 5327 1382 25.9 20070602 5008 3510 70.1

HPLC data Peak Area Peak Fresh solution Final solution Fresh solutionFinal solution No. of 20070601 of 20070601 of 20070602 of 20070602 1763.1 125.7 717.9 503.1 2 803.6 117.5 753.7 515.0 3 454.4 222.0 428.7320.1 4 588.1 74.6 554.8 366.7 5 516.2 217.6 479.1 347.4 6 212.5 63.7216.8 154.6 7 314.9 94.6 295.7 220.1 8 494.3 117.5 460.6 321.8 9 52.817.6 49.5 33.7 10 138.1 21.4 127.6 86.4 11 209.7 66.2 196.1 138.6 12179.5 63.7 168.3 119.0 13 26.3 4.1 24.0 16.3 14 477.0 154.7 445.3 305.715 96.5 20.6 90.1 61.5 Total 5327 1382 5008 3510

Peak identification of HPLC chromatograms Peak No anthocyanoside 1Delphinidin 3-o-galactoside 2 Delphinidin 3-o-glucoside 3 Cyanidin2-o-galactoside 4 Delphinidin 3-o-arabinoside 5 Cyanidin 3-o-glucoside 6Petunidin 3-o-galactoside 7 Cyanidin 3-o-arabinoside 8 Petunidin3-o-glucoside 9 Peonidin 3-o-galactoside 10 Petunidin 3-o-arabinoside 11Peonidin 3-o-glucoside 12 Malvidin 3-o-galactoside 13 Peonidin3-o-arabinoside 14 Malvidin 3-o-arabinoside 15 Malvidin 3-o-glucoside

Methods

Culturing of CaCo-2 Cells

CaCo-2 cells were cultured in Dulbeccos's Modified Eagle Mediumcontaining 20% fetal bovine serum, 1.2% nonessential amino acids, 0.83mM L-glutamine, 1.2% penicillin-streptomycin and 0.1% mercaptoethanol inan atmosphere of 5% CO₂ and 95% air at 37° C.

Cells were grown in 75 cm² culture-flasks (T75) and subcultured afterone week (every other day washed with PBS buffer, removed with trypsinand transferred to an new culture flask).

CaCo-2 Test

For experiments, cells were seeded in 6 well plates at a density of3×10⁵ cells per well and grown in an atmosphere of 5% CO₂ and 95% air at37° C. 7 to 8 days until confluency was reached. The cells were washedwith PBS buffer, incubated with 4 ml medium containing bilberry (30-60mg/100 ml medium) or black currant extract (30-60 mg/100 ml medium) for30, 60 or 120 minutes. For stabilizing experiments, medium containing 30mg glutathione/100 ml was used.

After the corresponding incubation time, 900 μl of incubation mediumused was taken from each well and mixed with 100 μl formic acid. Thecells were washed with PBS buffer and removed using 1 ml of 10% formicacid. Cells were sonicated 3 times for 30 seconds, centrifuged for 10minutes and the pellets were discarded. The supernatant was used assample for HPLC.

For comparison, the stability of anthocyanosides was tested inincubation medium without cells at 37° C. between 0 and 120 min.Incubation medium was stabilized with formic acid as described above.

HPLC Analytical Method (Materials, Instruments and Methods):

Acetonitrile, Methanol (HPLC grade), Formic acid (AR), Distilled water.Reference standard: Cyanidin-3-O-glucoside (Cl salt, Item 1201,Polyphenols Laboratories AS, Norway)

Pump: Merck Quaternary Gradient pump 6200 Autosampler: Merck AS 2000,Detector: HP-MVD 1050 set to 520 nm Column: Bischoff, Hypersil ODS, 250× 4.6 mm Mobile phase: A: formic acid/water = 10/90 B:methanol/acetonitrile/formic acid/water = 20/20/10/50 Gradient profile:(See FIG. 18) Flow rate: 1.5 ml/min Injection volume: 20 μl Temperature:45° C. Detection: 520 nm Calibration 5-point calibration withcyaniding-3-O- glucoside Quantitation: External standardization based onlinear regression analysis. The response factors of all anthocyanosidesseparated is quantitified against cyanidin-3-O-glucoside.

Standard Preparation (Cyanidin-3-O-glucoside)

Transfer an appropriate amount of standard into a 10 ml flask anddissolve in 1 ml MeOH. The flask was filled to volume with 10%phosphoric acid. Dilutions were prepared in 10% phosphoric acid.

Sample Preparation

Test solutions from incubation experiments (with/without cells andwith/without stabilizing agents) were used for analysis afterstabilization of anthocyanosides with formic acid. Samples were clearedby filtration prior to injection on to HPLC-system.

For example, 0.500 g Bilberry extract was dissolved in 10 ml methanolwith 2% HCl and ultrasonicated for 2 minutes. 1 ml of solution wastransferred into a 10 ml flask and diluted to the volume with 10%phosphoric acid. Sample was injected onto the HPLC-system.

Data Evaluation

% degradation is given in % of the initial values obtained.

% uptake into cells refers to the ratio of the amounts obtained in theincubation medium at the corresponding time to the amount found in thecells.

Stabilized Anthocyanins at Various pH Values

Raw Materials

Bilberry Extract, 20070602, Omnica GmbH

Reduced L-glutathione, Bio-Chemical reagent

Sodium phosphate buffer, pH values from 3.0 to 11.0

20 mg reduced L-glutathione were added to 100 ml flasks with 60 ml 5%sodium phosphate buffer (for appropriate pH). After the solid wastotally dissolved, 60 mg bilberry extract was added with stirring andput into water bath at 37° C. Samples from the various pH bufferedsolutions were withdrawn and analyzed by HPLC by the methods describedabove.

EXAMPLE 12

Product (Bilberry extract, Omnica GmbH (Hamburg) was adjusted to 37%(m/m) anthocyanosides. Based on an average content of 0.3-0.4% (m/m) ofanthocyanosides in fresh bilberries, the extract at hand represented a100:1 concentrate. The remaining 63% of the extract are represented inthe majority by proteins and carbohydrates occurring in fresh berries.500 mg bilberry extract and 500 mg bilberry extract with 50 mgL-Cysteine were combined to produce the bilberry/cysteine combo utilizedbelow. Bilberry extract noted below is the product described abovewithout the addition of cysteine.

12 volunteers received bilberry extract for 7 consecutive days. After a1 week wash-out period the same volunteers received Bilberry/Cysteinecombo for 7 consecutive days.

The design was a randomized, cross-over observation.

Plasma samples were taken during each period on day 1 (prior to and 0.5,1.5, 2.5 and 4 hours after treatment), 4 (prior to and 0.5 hours aftertreatment) and 7 ((prior to and 0.5, 1.5, 2.5 and 4 hours aftertreatment) of the corresponding treatment.

The main target of the study was to determine the plasma levels of theanthocyanins present in both formulations.

In a first run of analysis, all anthocyanins present in plasma wereextracted by solid phase extraction and after collection hydrolyzed toyield the anthocyanidins. This procedure was chosen to provideinformation on the real absorption of bilberry anthocyanins from bothformulations and to avoid erroneous conclusions due to metabolism of thecompounds absorbed. Each metabolite (i.e. glucuronides) would not countas absorbed from the formulations although the efficacy resides in theanthocyanidin skeleton and not in a specific glycosidation pattern.

The analysis comprised HPLC with UV-detection at 540 nm. Quantificationwas based on external standardization with authentic anthocyanidinstandards. The recovery of the method was estimated withdelphinidin-3-O-glucoside and cyanidin-3-O-glucoside. These two bilberryanthocyanins were spiked to blank plasma, extracted and hydrolyzed inthe same way as the study samples. It was shown that the recoveryis >95%. The stability of the study samples was confirmed by spikedblank plasma samples treated and stored exactly the same ways as thestudy samples.

Pharmacokinetic parameters were calculated using standardnon-compartmental methods.

LIST OF ABBREVIATIONS ANOVA Analysis of Variances Ara Arabinose AUC Areaunder the Curve AUC_((0-inf)) AUC from time-point 0 to infinity.AUC_((0-t)) AUC from time-point 0 to the last measured time-point.AU_((0-4h)) Area under the Curve from time-point 0 to 8 hours. βElimination rate constant C_(max) Maximum plasma concentration obtainedCn Cyanidin CV Coefficient of Variation Dp Delphinidin Gal Galactose GlcGlucose HPLC High Performance Liquid Chromatography MS Mass SpectrometryMv Malvidin Pe Peonidin Pt Petunidin t_(1/2) Terminal plasma half-lifeT_(max) Time to maximum plasma concentration (C_(max)) UV Ultra-Violet

Study Results:

FIGS. 27 through 40 demonstrate that anthocyanidin concentration ofbilberry/cysteine combinations is increased by at least twice the amountof anthocyanidin concentration in blood plasma when compared to samplesfrom bilberry without the addition of cysteine. This can be seen intotal anthocyanidin content as well as individual anthocyanidins.

The following tables provide the mean values ± standard deviation (s.d.)of the plasma levels determined for the 5 anthocyanidins present inbilberry (i.e. cyanidin [Cn], delphindin [Dp], petunidin [Pt], peonidin[Pe] and malvidin [Mv]).

TOTAL CYANIDIN PLASMA LEVELS (NG/ML), MEAN ± S.D. Total Cn (ng/mL)Bilberry/Cysteine Bilberry combo Time (h) Mean s.d. Mean s.d Day 1 0 BLDn.a. BLD n.a. 0.5 22.3 7.2 35.7 8.6 1.5 18.4 5.7 27.2 8.3 2.5 9.1 4.114.5 5.8 4 3.2 1.9 5.2 2.4 Day 4 5 BLD n.a. BLD n.a. 5.5 17.9 6   30.16.8 Day 7 6 BLD n.a. BLD n.a. 6.5 26.3 8.6 51.7 12.4  7.5 23.9 6.7 29.59.6 8.5 14.6 5.4 25.6 7.5 10 5.9 2.7 7.8 3.2 BLD . . . BELOW LIMIT OFDETERMINATION (<1 NG/ML), N.A. . . . NOT APPLICABLE

TOTAL DELPHINIDIN PLASMA LEVELS (NG/ML), MEAN ± S.D. Total Dp (ng/mL)Bilberry/Cysteine Bilberry combo Time (h) Mean s.d. Mean s.d Day 1 0 BLDn.a. BLD n.a. 0.5 18.6 6.9 29.2 9.3 1.5 12.4 6.2 20.7 7.4 2.5 6.2 3.112.5 5.3 4 3.9 1.8 5.1 2.6 Day 4 0 BLD n.a. BLD n.a. 0.5 19.4 7.4 32.611.5  Day 7 0 BLD n.a. BLD n.a. 0.5 27.4 8.5 53.7 24.8  1.5 14.9 6.834.9 13.8  2.5 7.1 4.5 16.3 8.2 4 4.2 3.5 6.8 3.7

TOTAL PETUNIDIN PLASMA LEVELS (NG/ML), MEAN ± S.D. Total Pt (ng/mL)Bilberry/Cysteine Bilberry combo Time (h) Mean s.d. Mean s.d Day 1 0 BLDn.a. BLD n.a. 0.5 10.2 4.5 19.8 7.1 1.5 5.8 3.7 8.9 4.5 2.5 3.1 2.3 5.22.3 4 1.2 1.6 1.9 1.6 Day 4 0 BLD n.a. BLD n.a. 0.5 9.2 4.2 16.8 7.6 Day7 0 BLD n.a. BLD n.a. 0.5 14.4 6.7 29.8 12.6  1.5 8.1 4.8 14.3 6.4 2.53.7 1.9 6.2 3.7 4 1.6 1.5 2.9 1.5

TOTAL MALVIDIN PLASMA LEVELS (NG/ML), MEAN ± S.D. Total Mv (ng/mL)Bilberry/Cysteine Bilberry combo Time (h) Mean s.d. Mean s.d Day 1 0 BLDn.a. BLD n.a. 0.5 10.9 5.4 21.1 9.6 1.5 5.2 3.1 11.2 5.8 2.5 2.1 1.5 5.82.6 4 1.2 1   2.3 1.4 Day 4 0 BLD n.a. BLD n.a. 0.5 12.5 6.3 21.7 11.4 Day 7 0 BLD n.a. BLD n.a. 0.5 14.3 6.8 34.5 16.4  1.5 6.4 3.2 18.5 7.92.5 3.8 1.8 9.6 4.4 4 1.6 1.2 3.1 1.9

TOTAL PEONIDIN PLASMA LEVELS (NG/ML), MEAN ± S.D. Total Pe (ng/mL)Bilberry/Cysteine Bilberry combo Time (h) Mean s.d. Mean s.d Day 1 0 BLDn.a. BLD n.a. 0.5 4.0 2.3 7.9 3.6 1.5 2.2 1.9 3.2 2.1 2.5 1   1.1 1.81   4 BLD n.a. 1   n.a. Day 4 0 BLD n.a. BLD n.a. 0.5 4.6 2.7 8.2 4.4Day 7 0 BLD n.a. BLD n.a. 0.5 6.3 3.0 12.2  5.3 1.5 2.7 1.5 7.5 3.7 2.51.4 0.8 3.8 1.9 4 BLD n.a. 1.4 1.1

The following Tables provide the pharmacokinetic parameters calculatedfrom the mean plasma levels observed.

PHARMACOKINETIC PARAMETERS CALCULATED FOR TOTAL CYANIDIN Day 1 Day 7Bilberry/ Bilberry/ Cysteine Cysteine Parameter Bilberry combo Bilberrycombo C_(max) (ng/mL) 22.3 35.7 26.3 51.7 T_(max) (h) 0.5 0.5 0.5 0.5AUC_(0-t) (ng × h/mL) 48.9 76 66.3 106.1 t_(1/2) (h) 1.20 1.23 1.56 1.34AUC_(0-inf) (ng × h/mL) 64.7 101.7 99.2 155.6

PHARMACOKINETIC PARAMETERS CALCULATED FOR TOTAL DELPHINIDIN Day 1 Day 7Bilberry/ Bilberry/ Cysteine Cysteine Parameter Bilberry combo Bilberrycombo C_(max) (ng/mL) 18.6 29.2 27.4 53.7 T_(max) (h) 0.5 0.5 0.5 0.5AUC_(0-t) (ng × h/mL) 37.0 62.1 47.5 100.7 t_(1/2) (h) 1.5 1.4 1.3 1.1AUC_(0-inf) (ng × h/mL) 50.5 86.8 60.5 127.5

PHARMACOKINETIC PARAMETERS CALCULATED FOR TOTAL PETUNIDIN Day 1 Day 7Bilberry/ Bilberry/ Cysteine Cysteine Parameter Bilberry combo Bilberrycombo C_(max) (ng/mL) 10.2 19.8 14.4 29.8 T_(max) (h) 0.5 0.5 0.5 0.5AUC_(0-t) (ng × h/mL) 18.2 31.7 24.7 46.6 t_(1/2) (h) 0.6 0.7 0.6 0.7AUC_(0-inf) (ng × h/mL) 1.1 1.1 1.1 1.0

PHARMACOKINETIC PARAMETERS CALCULATED FOR TOTAL PEONIDIN Day 1 Day 7Bilberry/ Bilberry/ Cysteine Cysteine Parameter Bilberry combo Bilberrycombo C_(max) (ng/mL) 4.0 7.9 6.3 12.2 T_(max) (h) 0.5 0.5 0.5 0.5AUC_(0-t) (ng × h/mL) 6.5 12.1 9.2 22.5 t_(1/2) (h) 1.0 1.2 0.9 1.1AUC_(0-inf) (ng × h/mL) 7.9 15.2 11.0 28.5

PHARMACOKINETIC PARAMETERS CALCULATED FOR TOTAL MALVIDIN Day 1 Day 7Bilberry/ Bilberry/ Cysteine Cysteine Parameter Bilberry combo Bilberrycombo C_(max) (ng/mL) 10.9 21.1 14.3 34.5 T_(max) (h) 0.5 0.5 0.5 0.5AUC_(0-t) (ng × h/mL) 16.9 36.0 23.1 58.7 t_(1/2) (h) 1.1 1.1 1.1 1.0AUC_(0-inf) (ng × h/mL) 20.2 45.1 29.3 72.6

EXAMPLE 13 Sample Preparation

Sample A was the same as the blank sample of example 1.

Sample B: 48 mg (9.5% by weight of reduced glutathione) beer yeastextract was added to a 100 ml flask with 60.0 ml 5% Sodium phosphatebuffer (pH=7.0). The solution was stirred until homogeneous and then 60mg (0.049 mmol of anthocyanins) bilberry extract was added with stirringuntil uniform. The sample was placed into a 37° C. water bath for 4hours with stirring. Degradation ratio analysis was monitored by HPLC.

Sample C: The preparation was the same as example B with the provisothat the amount of beer yeast extract was 120 mg.

Results:

Sample A B C Residue ratio 22.1% 50.9% 77.9%

HPLC test parameters were the same as the above example.

The present example represents a positive protective effect of beeryeast extract on diminishing the degradation of the anthocyanin contentof the samples.

Although the present invention has been described with reference topreferred embodiments, persons skilled in the art will recognize thatchanges may be made in form and detail without departing from the spiritand scope of the invention. All references cited throughout thespecification, including those in the background, are incorporatedherein in their entirety.

Those skilled in the art will recognize, or be able to ascertain, usingno more than routine experimentation, many equivalents to specificembodiments of the invention described specifically herein. Suchequivalents are intended to be encompassed in the scope of the followingclaims.

1. A stabilized anthocyanin extract composition consisting of a bilberryextract and cysteine.
 2. The stabilized anthocyanin extract compositionof claim 1, wherein the mole ratio of cysteine to bilberry extract isabout 0.1 to about
 10. 3. The stabilized anthocyanin extract compositionof claim 1, wherein the composition is stabile toward degradation whenexposed to an aqueous environment with a pH of between about 2 and about12.